Microfluidic Protein Crystallography Techniques

ABSTRACT

The present invention relates to microfluidic devices and methods facilitating the growth and analysis of crystallized materials such as proteins. In accordance with one embodiment, a crystal growth architecture is separated by a permeable membrane from an adjacent well having a much larger volume. The well may be configured to contain a fluid having an identity and concentration similar to the solvent and crystallizing agent employed in crystal growth, with diffusion across the membrane stabilizing that process. Alternatively, the well may be configured to contain a fluid having an identity calculated to affect the crystallization process. In accordance with the still other embodiment, the well may be configured to contain a material such as a cryo-protectant, which is useful in protecting the crystalline material once formed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/748,838, filed May 15, 2007, which is a continuation of U.S. patentapplication Ser. No. 10/810,350, filed Mar. 26, 2004, which is acontinuation-in-part of U.S. nonprovisional patent application no.10/637,847, filed Aug. 7, 2003, which is a continuation-in-part of U.S.nonprovisional patent application Ser. No. 10/117,978, filed Apr. 5,2002, which claims priority as a nonprovisional application from U.S.provisional patent application No. 60/323,524 filed Sep. 17, 2001, andwhich is also a continuation-in-part of U.S. nonprovisional applicationSer. No. 09/887,997 filed Jun. 22, 2001, which is in turn acontinuation-in-part of U.S. nonprovisional patent application Ser. No.09/826,583 filed Apr. 6, 2001. U.S. patent application Ser. No.10/810,350, filed Mar. 26, 2004 is also a continuation-in-part of U.S.nonprovisional patent application Ser. No. 10/265,473, filed Oct. 4,2002, which further claims priority from U.S. provisional patentapplication No. 60/527,168 filed Dec. 5, 2003, and from U.S. provisionalpatent application No. 60/527,625 filed Dec. 5, 2003. These prior patentapplications are hereby incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Work described herein has been supported, in part, by NSF (xyz in a chipprogram); National Institute of Health grant CA 77373; NSERC (JuliePayette Fellowship); David H. & Lucille M. Packard Foundation; and G.Harold and Leila Y. Mathers Charitable Foundation. The United StatesGovernment may therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

Crystallization is an important technique to the biological and chemicalarts. Specifically, a high-quality crystal of a target compound can beanalyzed by x-ray diffraction techniques to produce an accuratethree-dimensional structure of the target. This three-dimensionalstructure information can then be utilized to predict functionality andbehavior of the target.

In theory, the crystallization process is simple. A target compound inpure form is dissolved in solvent. The chemical environment of thedissolved target material is then altered such that the target is lesssoluble and reverts to the solid phase in crystalline form. This changein chemical environment typically accomplished by introducing acrystallizing agent that makes the target material is less soluble,although changes in temperature and pressure can also influencesolubility of the target material.

In practice however, forming a high quality crystal is generallydifficult and sometimes impossible, requiring much trial and error andpatience on the part of the researcher. Specifically, the highly complexstructure of even simple biological compounds means that they are notamenable to forming a highly ordered crystalline structure. Therefore, aresearcher must be patient and methodical, experimenting with a largenumber of conditions for crystallization, altering parameters such assample concentration, solvent type, countersolvent type, temperature,and duration in order to obtain a high quality crystal, if in fact acrystal can be obtained at all.

Accordingly, there is a need in the art for methods and structures forperforming high throughput screening of crystallization of targetmaterials.

SUMMARY OF THE INVENTION

The present invention relates to microfluidic devices and methodsfacilitating the growth and analysis of crystallized materials such asproteins. In accordance with one embodiment, a crystal growtharchitecture is separated by a permeable membrane from an adjacent wellhaving a much larger volume. The well may be configured to contain afluid having an identity and concentration similar to the solvent andcrystallizing agent employed in crystal growth, with diffusion acrossthe membrane stabilizing that process. Alternatively, the well may beconfigured to contain a fluid having an identity calculated to affectthe crystallization process. In accordance with the still otherembodiment, the well may be configured to contain a material such as acryo-protectant, which is useful in protecting the crystalline materialonce formed.

An embodiment of a method in accordance with the present invention forgrowing crystals, comprises, disposing a solution including a solventand containing a crystal source material in a first chamber defined byan elastomer structure. A crystallizing agent is disposed in a secondchamber defined by the elastomer structure. A material is disposed in awell defined by the elastomer structure adjacent to and separated fromthe first and second chambers by an elastomer membrane. The first andsecond chambers are placed in fluid communication to alter a solubilityof the crystal source material, such that a presence of the materialaffects formation of a crystal from the crystal source material.

An embodiment of apparatus in accordance with the present invention forforming crystals, comprises, an elastomer structure defining a firstchamber in selective fluid communication with a second chamber. Thefirst chamber is configured to contain a solution containing a crystalmaterial dissolved in a solvent, and the second chamber is configured tocontain a crystallizing agent, the first and second chambers separatedfrom an adjacent well by a thin elastomer membrane.

An embodiment of an apparatus in accordance with the present inventionfor extracting a crystal from an elastomer microfluidic device,comprises, an enclosure and a piston slidable within the enclosure andcomprising an ejector portion having pins moveable relative to a bladeportion having blades. A loose spring is positioned between the bladeportion and the enclosure and configured to bias the piston. A tightspring is positioned between the blade portion and the ejector portionand configured to bias the ejector portion relative to the bladeportion.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17 shows a CAD drawing of a large-capacity embodiment of a crystalgrowth chip in accordance with the present invention.

FIG. 18 shows a micrograph of a protein crystal grown utilizing thedevice of FIG. 17.

FIG. 19A is a simplified cross-sectional schematic view of an embodimentof a crystal growth/analysis device in accordance with the presentinvention.

FIG. 19B is an enlarged micrograph showing a perspective view of theembodiment of a crystal growth/analysis device shown in FIG. 19A.

FIG. 19C is an enlarged micrograph showing a plan view of the embodimentof a crystal growth/analysis device shown in FIGS. 19A-B.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIG. 21 is an enlarged cross-sectional view of the inverted membrane ofthe device shown in FIG. 19.

FIG. 22 is a micrograph showing a plan view of a crystal disk inaccordance with an embodiment of the present invention.

FIG. 23 is a micrograph showing a crystal disk in accordance with anembodiment of the present invention held in a mounting clip.

FIG. 24 shows protein crystals grown utilizing the design of FIG. 21.

FIG. 25 shows a high resolution diffraction from a protein crystalsgrown, frozen and harvested utilizing the design of FIG. 21.

FIGS. 26A-26D show plan views illustrating operation of one embodimentof a cell pen structure in accordance with the present invention.

FIGS. 27A-27B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 28A-28D show plan views of operation of a structure utilizingcross-channel injection in accordance with the embodiment of the presentinvention.

FIGS. 29A-D are simplified schematic diagrams plotting concentrationversus distance for two fluids in diffusing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIGS. 30A-B show simplified cross-sectional views of the attemptedformation of a macroscopic free-interface in a capillary tube.

FIGS. 31A-B show simplified cross-sectional views of convective mixingbetween a first solution and a second solution in a capillary tuberesulting from a parabolic velocity distribution of pressure drivenPoiseuille flow

FIGS. 32A-C show simplified cross-sectional views of interaction in acapillary tube between a first solution having a density greater thanthe density of second solution.

FIG. 33A shows a simplified cross-sectional view of a microfluidic freeinterface in accordance with an embodiment of the present invention.

FIG. 33B shows a simplified cross-sectional view of a conventionalnon-microfluidic interface.

FIGS. 34A-D show plan views of the priming of a flow channel andformation of a microfluidic free interface in accordance with anembodiment of the present invention.

FIGS. 35A-E show simplified schematic views of the use of“break-through” valves to create a microfluidic free interface.

FIG. 36A shows a simplified schematic view of a protein crystal beingformed utilizing a conventional macroscopic free interface diffusiontechnique.

FIG. 36B shows a simplified schematic view of a protein crystal beingformed utilizing diffusion across a microfluidic free interface inaccordance with an embodiment of the present invention.

FIG. 37A shows a simplified plan view of a flow channel overlapped atintervals by a forked control channel to define a plurality of chambers(A-G) positioned on either side of a separately-actuated interfacevalve.

FIGS. 37B-D plot solvent concentration at different times for the flowchannel shown in FIG. 37A.

FIG. 38A shows three sets of pairs of chambers connected bymicrochannels of a different length.

FIG. 38B plots equilibration time versus channel length.

FIG. 39 shows four pairs of chambers, each having different arrangementsof connecting microchannel(s).

FIG. 40A shows a plan view of a simple embodiment of a microfluidicstructure in accordance with the present invention.

FIG. 40B is a simplified plot of concentration versus distance for thestructure of FIG. 40A.

FIG. 41 plots the time required for the concentration in one of thereservoirs of FIG. 40 to reach 0.6 of the final equilibrationconcentration, versus channel length.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interface ofFIG. 40.

FIG. 43 presents a phase diagram depicting the phase space betweenfluids A and B, and the path in phase space traversed in the reservoirsas the fluids diffuse across the microfluidic free interface of FIG. 40

FIG. 44 shows an enlarged view of one embodiment of a chip holder inaccordance with the present invention.

FIG. 45A shows a simplified plan view of the alternative embodiment ofthe chip utilized to obtain experimental results.

FIG. 45B shows as simplified enlarged plan view of a set of threecompound wells of the chip shown in FIG. 45A.

FIG. 45C shows a simplified cross-sectional view of the compound wellsof FIGS. 45A-B.

FIG. 46 shows a plan view of the creation of a microfluidic freeinterface between a flowing fluid and a dead-ended branch channel.

FIG. 47 shows a simplified plan view of one embodiment of a microfluidicstructure in accordance with the present invention for creatingdiffusion gradients of two different species in different dimensions.

FIG. 48 shows a simplified plan view of an alternative embodiment of amicrofluidic structure in accordance with the present invention forcreating diffusion gradients of two different species in differentdimensions.

FIG. 49 shows a simplified plan view of a sorting device in accordancewith an embodiment of the present invention.

FIG. 50 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention.

FIG. 51 plots injected volume versus injection cycles during operationof one embodiment of a cross-flow injection structure in accordance withthe present invention.

FIG. 52 plots crystallization hits utilizing a microfluidic chip inaccordance with the present invention.

FIGS. 53A-B show plan and cross-sectional views of one embodiment of acrystal growing/harvesting chip in accordance with of one embodiment ofthe present invention.

FIG. 54A shows a plan view of one embodiment of a combinatoricmixing/storage structure in accordance with the present invention.

FIG. 54B shows an enlarged view of the mixing portion of the structureof FIG. 54A.

FIG. 54C shows an enlarged view of the storage array of the structure ofFIG. 54A.

FIG. 54D shows an enlarged view of one cell of the storage array of thestructure of FIG. 54C.

FIG. 55 shows a simplified cross-sectional view of one embodiment of atool which allows the removal of crystal-containing portions of a chipfor analysis.

FIG. 56 shows a simplified enlarged view of the tool of FIG. 55.

FIG. 57 plots trajectory through the phase space achieved by a number ofcrystallization approaches.

FIG. 58A-D illustrates a schematic view of storage technique utilizing agated serpentine storage line.

FIG. 59 illustrates a schematic view of an embodiment wherein amultiplexer could be used to direct each experimental condition intoparallel storage channels.

FIG. 60 illustrates a schematic view of an embodiment wherein eachreaction condition is dead-end filled to the end of a storage line.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No.09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filed Jun. 27,2000. These patent applications are hereby incorporated by reference.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogeneous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogeneous aspect, the bonding process usedto bind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any clastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(Styrene-Butadiene-Styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves.

Smaller and larger valves and switching valves are contemplated in thepresent invention, and a non-exclusive list of ranges of dead volumeincludes 1 aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL,and 1 pL to 100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:

w=(BPb ⁴)/(Eh ³), where:  (1)

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τclose is expected to be smallerthan τopen. There is also a lag between the control signal and controlpressure response, due to the limitations of the miniature valve used tocontrol the pressure. Calling such lags t and the 1/e time constants τ,the values are: topen=3.63 ms, τopen=1.88 ms, tclose=2.15 ms,τclose=0.51 ms. If 3τ each are allowed for opening and closing, thevalve runs comfortably at 75 Hz when filled with aqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH 8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log2n) controllines.

8. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure foamed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIGS. 20A-D allows a switchable flow array tobe constructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

9. Cell Pen/Cell Cage

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 26A-26D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 26A has been loaded with cells A-H that havebeen previously sorted. FIGS. 26B-26C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 26D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a.

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.27A and 27B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 26A-26D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

The cross-flow channel architecture illustrated shown in FIGS. 26A-26Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 28A-E, which illustrate a plan view of mixingsteps performed by a microfabricated structures in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 28A, valve pair 7408 c-d is initially opened whilevalve pair 7408 a-b is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7404. Valve pair 7408 a-b is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 28B, valve pairs 7408 c-d are closed and 7408 a-bare opened, such that fluid sample 7410 is injected from intersection7412 into flow channel 7402 bearing a cross-flow of fluid. The processshown in FIGS. 28A-B can be repeated to accurately dispense any numberof fluid samples down cross-flow channel 7402.

While the embodiment of a process-channel flow injector structure shownin FIGS. 28A-B feature channels intersecting at a single junction, thisis not required by the present invention. Thus FIG. 28C shows asimplified plan view of another embodiment of an injection structure inaccordance with the present invention, wherein junction 7450 betweenintersecting flow channels 7452 is extended to provide additional volumecapacity. FIG. 28D shows a simplified plan view of yet anotherembodiment of an injection structure in accordance with the presentinvention, wherein elongated junction 7460 between intersecting flowchannels 7462 includes branches 7464 to provide still more injectionvolume capacity.

And while the embodiment shown and described above in connection withFIGS. 28A-28D utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

FIG. 50 plots Log(R/B) vs. number of slugs injected for one embodimentof a cross-flow injection system in accordance with the presentinvention. The reproducibility and relative independence of metering bycross-flow injection from process parameters such as flow resistance isfurther evidenced by FIG. 51, which plots injected volume versus numberof injection cycles for cross-channel flow injection under a variety offlow conditions. FIG. 51 shows that volumes metered by cross-flowinjection techniques increase on a linear basis over a succession ofinjection cycles. This linear relationship between volume and number ofinjection cycles is relatively independent of flow resistance parameterssuch as elevated fluid viscosity (imparted by adding 25% glycerol) andthe length of the flow channel (1.0-2.5 cm).

II. Crystallization Structures and Methods

High throughput screening of crystallization of a target material, orpurification of small samples of target material by recrystallization,may be accomplished by simultaneously introducing a solution of thetarget material at known concentrations into a plurality of chambers ofa microfabricated fluidic device. The microfabricated fluidic device isthen manipulated to vary solution conditions in the chambers, therebysimultaneously providing a large number of crystallization environments.Control over changed solvent conditions may result from a variety oftechniques, including but not limited to metering of volumes of acrystallizing agent into the chamber by volume exclusion, by entrapmentof liquid volumes determined by the dimensions of the microfabricatedstructure, or by cross-channel injection into a matrix of junctionsdefined by intersecting orthogonal flow channels.

Crystals resulting from crystallization in accordance with embodimentsof the present invention can be utilized for x-ray crystallography todetermine three-dimensional molecular structure. Alternatively, wherehigh throughput screening in accordance with embodiments of the presentinvention does not produce crystals of sufficient size for direct x-raycrystallography, the crystals can be utilized as seed crystals forfurther crystallization experiments. Promising screening results canalso be utilized as a basis for further screening focusing on a narrowerspectrum of crystallization conditions, in a manner analogous to the useof standardized sparse matrix techniques.

Systems and methods in accordance with embodiments of the presentinvention are particularly suited to crystallizing larger biologicalmacromolecules or aggregates thereof, such as proteins, nucleic acids,viruses, and protein/ligand complexes. However, crystallization inaccordance with the present invention is not limited to any particulartype of target material.

As employed in the following discussion, the term “crystallizing agent”describes a substance that is introduced to a solution of targetmaterial to lessen solubility of the target material and thereby inducecrystal formation. Crystallizing agents typically includecountersolvents in which the target exhibits reduced solubility, but mayalso describe materials affecting solution pH or materials such aspolyethylene glycol that effectively reduce the volume of solventavailable to the target material. The term “countersolvent” is usedinterchangeably with “crystallizing agent”.

1. Crystallization by Volume Entrapment

FIG. 45A shows a simplified plan view of an embodiment of acrystallization system wherein metering of different volumes ofcountersolvent is determined by photolithography during formation of theflow channels. FIG. 45B shows a simplified enlarged plan view of a setof three compound wells of the device of FIG. 45A. FIG. 45C shows asimplified cross-sectional view of the wells of FIG. 45B along lineC-C′. This chip design employed metering of target solution andcrystallizing agent utilizing the volume entrapment technique.

Specifically, each chip 9100 contains three compound wells 9102 for eachof the 48 different screen conditions, for a total of 144 assays perchip. A compound well 9102 consists of two adjacent wells 9102 a and9102 b etched in a glass substrate 9104, and in fluidic contact via amicrochannel 9106 In each of the compound wells 9102, the proteinsolution is combined with the screen solution at a ratio that is definedby the relative size of the adjacent wells 9102 a-b. In the particularembodiment shown in FIGS. 45A-C, the three ratios were(protein:solution) 4:1, 1:1, and 1:4. The total volume of each assay,including screen solution, is approximately 25 nL. However, the presentinvention is not limited to any particular volume or range of volumes.Alternative embodiments in accordance with the present invention mayutilize total assay volumes of less than 10 nL, less than 5 nL, lessthan 2.5 nL, less than 1.0 nL, and less than 0.5 nL.

The chip control layer 9106 includes an interface control line 9108, acontainment control line 9110 and two safety control lines 9112. Controllines 9108, 9110, and 9112 are filled with water rather than air inorder to maintain a humid environment within the chip and to preventdehydration of the flow channels and chambers in which crystallizationis to be performed.

The interface valves 9114 bisect the compound wells 9102, separating theprotein from the screen until completion of loading. Containment valves9116 block the ports of each compound well 9102, isolating eachcondition for the duration of the experiment. The two safety valves 9118are actuated during protein loading, and prevent spillage of proteinsolution in the event of a failed interface valve.

Fabrication of the microfluidic devices utilized in the experiments wereprepared by standard multilayer soft lithography techniques and sealedto an etched glass slide by baking at 80° C. for 5 hours or greater. Theglass substrate is masked with a 16 um layer of 5740 photoresist, and ispatterned using standard photolithography. The glass substrate is thenetched in a solution of 1:1:1 (BOE:H₂O:2N HCl) for 60 minutes, creatingmicro-wells with a maximum depth of approximately 80 μm.

The chip fabrication protocol just described is only one example of apossible embodiment of the present invention. In accordance withalternative embodiments, the crystallization chambers and flow channelscould be defined between a planar substrate and a pattern of recessesformed entirely in the lower surface of the elastomer portion. Stillfurther alternatively, the crystallization chambers and flow channelscould be defined between a planar, featureless lower surface of theelastomer portion and a pattern of recesses formed entirely in thesubstrate.

Crystallization on chip is set up as follows. All control lines in chipcontrol layer 9106 are loaded with water at a pressure of 15-17 psi.Once the control lines are filled and valves 9114 and 9116 arecompletely actuated, the containment valve 9116 is released, and proteinis loaded through the center via 9120 using about 5-7 psi. The proteinsolution completely fills the protein side of each compound well 9102.Failed valves, if present, are then identified, and vacuum grease isplaced over the corresponding screen via to prevent subsequentpressurization, and possible contamination of the remaining conditions.2.5 to 4 μL of a sparse matrix screen (typically Hampton Crystal ScreenI, 1-48) are then pipetted into the screen vias 9122. The safety valves9118 are released, and a specially designed chip holder (describedbelow) is used to create a pressurized (5-7 psi) seal over all 48 screenvias 9122. The screen solutions are dead end loaded, filling the screenside of each compound well. Protein and crystal screen reagents are keptseparate with the interface valve until all wells are loaded, at whichpoint the containment valve is closed and the interface valve opened toallow diffusion between liquid volumes present in the two halves of thecompound wells 9102.

For these experiments, the average time spent setting up an experiment,including filling control lines, was approximately 35 min, with thefastest experiment taking only 20 minutes to set up. This set up timecould potentially be reduced even further through the use of roboticpipetting of solutions to the chip, or through the use of pressures toload and prime delivered solutions, or through use of a microfluidicmetering device, for example the combinatorial mixing structurepreviously described.

As previously illustrated, embodiments of microfluidic devices inaccordance with the present invention may utilize on-chip reservoirs orwells. However, in a microfluidic device requiring the loading of alarge number of solutions, the use of a corresponding large number ofinput tubes with separate pins for interfacing each well may beimpractical given the relatively small dimensions of the fluidic device.In addition, the automated use of pipettes for dispensing small volumesof liquid is known, and thus it therefore may prove easiest to utilizesuch techniques to pipette solutions directly on to wells present on theface of a chip.

Capillary action may not be sufficient to draw solutions from on-chipwells into active regions of the chip, particularly where dead-endedchambers are to be primed with material. In such embodiments, one way ofloading materials into the chip is through the use of externalpressurization. Again however, the small dimensions of the devicecoupled with a large number of possible material sources may renderimpractical the application of pressure to individual wells through pinsor tubing.

Accordingly, FIG. 44 shows an exploded view of a chip holder 11000 inaccordance with one embodiment of the present invention. Bottom portion11002 of chip holder 11000 includes raised peripheral portion 11004surrounding recessed area 11006 corresponding in size to the dimensionsof chip 11008, allowing microfluidic chip 11008 to be positionedtherein. Peripheral region 11004 further defines screw holes 11010.

Microfluidic device 11008 is positioned within recessed area 11006 ofbottom portion 11002 of chip holder 11000. Microfluidic device 11008comprises an active region 11011 that is in fluidic communication withperipheral wells 11012 configured in first and second rows 11012 a and11012 b, respectively. Wells 11012 hold sufficient volumes of materialto allow device 11008 to function. Wells 11012 may contain, for example,solutions of crystallizing agents, solutions of target materials, orother chemical reagents such as stains. Bottom portion 11002 contains awindow 11003 that enables active region 11011 of chip 11008 to beobserved.

Top portion 11014 of chip holder 11000 fits over bottom chip holderportion 11002 and microfluidic chip 11008 positioned therein. For easeof illustration, in FIG. 80 top chip holder portion 11014 is showninverted relative to its actual position in the assembly. Top chipholder portion 11014 includes screw holes 11010 aligned with screw holes11010 of lower holder portion 11002, such that screws 11016 may beinserted through holes 11010 secure chip between portions 11002 and11014 of holder 11000. Chip holder upper portion 11014 contains a window11005 that enables active region 11011 of chip 11008 to be observed.

Lower surface 11014 a of top holder portion 11014 includes raisedannular rings 11020 and 11022 surrounding recesses 11024 and 11026,respectively. When top portion 11014 of chip holder 11000 is pressedinto contact with chip 11008 utilizing screws 11016, rings 11020 and11022 press into the soft elastomeric material on the upper surface ofchip 11008, such that recess 11024 defines a first chamber over top row11012 a of wells 11012, and recess 11026 defines a second chamber overbottom row 11012 b of wells 11012. Holes 11030 and 11032 in the side oftop holder portion 11014 are in communication with recesses 11024 and11026 respectively, to enable a positive pressure to be applied to thechambers through pins 11034 inserted into holes 11030 and 11032,respectively. A positive pressure can thus simultaneously be applied toall wells within a row, obviating the need to utilize separateconnecting devices to each well.

In operation, solutions are pipetted into the wells 11012, and then chip11008 is placed into bottom portion 11002 of holder 11000. The topholder portion 11014 is placed over chip 11008, and is pressed down byscrews. Raised annular rings 11020 and 11022 on the lower surface of topholder portion 11014 make a seal with the upper surface of the chipwhere the wells are located. Solutions within the wells are exposed topositive pressures within the chamber, and are thereby pushed into theactive area of microfluidic device.

The downward pressure exerted by the chip holder may also pose theadvantage of preventing delamination of the chip from the substrateduring loading. This prevention of delamination may enable the use ofhigher priming pressures.

The chip holder shown in FIG. 44 represents only one possible embodimentof a structure in accordance with the present invention.

2. Control Over Other Factors Influencing Crystallization

While the above crystallization structures describe altering theenvironment of the target material through introduction of volumes of anappropriate crystallization agent, many other factors are relevant tocrystallization. Such additional factors include, but are not limitedto, temperature, pressure, concentration of target material in solution,equilibration dynamics, and the presence of seed materials.

In specific embodiments of the present invention, control overtemperature during crystallization may be accomplished utilizing acomposite elastomer/silicon structure previously described.Specifically, a Peltier temperature control structure may be fabricatedin an underlying silicon substrate, with the elastomer aligned to thesilicon such that a crystallization chamber is proximate to the Peltierdevice. Application of voltage of an appropriate polarity and magnitudeto the Peltier device may control the temperature of solvent andcountersolvent within the chamber.

Alternatively, as described by Wu et al. in “MEMS Flow Sensors forNano-fluidic Applications”, Sensors and Actuators A 89 152-158 (2001),crystallization chambers could be heated and cooled through theselective application of current to a micromachined resistor structureresulting in ohmic heating. Moreover, the temperature of crystallizationcould be detected by monitoring the resistance of the heater over time.The Wu et al. paper is hereby incorporated by reference for allpurposes.

It may also be useful to establish a temperature gradient across amicrofabricated elastomeric crystallization structure in accordance withthe present invention. Such a temperature gradient would subject targetmaterials to a broad spectrum of temperatures during crystallization,allowing for extremely precise determination of optimum temperatures forcrystallization.

With regard to controlling pressure during crystallization, embodimentsof the present invention employing metering of countersolvent by volumeexclusion are particularly advantageous. Specifically, once the chamberhas been charged with appropriate volumes of solvent and countersolvent,a chamber inlet valve may be maintained shut while the membraneoverlying the chamber is actuated, thereby causing pressure to increasein the chamber. Structures in accordance with the present inventionemploying techniques other than volume exclusion could exert pressurecontrol by including flow channels and associated membranes adjacent tothe crystallization chamber and specifically relegated to controllingpressure within the channel.

Another factor influencing crystallization is the amount of targetmaterial available in the solution. As a crystal forms, it acts as asink to target material available in solution, to the point where theamount of target material remaining in solution may be inadequate tosustain continued crystal growth. Therefore, in order to growsufficiently large crystals it may be necessary to provide additionaltarget material during the crystallization process.

Accordingly, the cell pen structure previously described in connectionwith FIGS. 27A-27B may be advantageously employed in crystallizationstructures in accordance with embodiments of the present invention toconfine growing crystals within a chamber. This obviates the danger ofwashing growing crystals down a flow channel that is providingadditional target material, causing the growing crystals to be lost inthe waste.

Moreover, the cell cage structure of FIGS. 27A-27B may also be usefulduring the process of crystal identification. Specifically, salts areoften present in the sample or countersolvent, and these salts may formcrystals during crystallization attempts. One popular method ofdistinguishing the growth of salt crystals from the target crystals ofinterest is through exposure to a staining dye such as IZIT™,manufactured by Hampton Research of Laguna Niguel, Calif. This IZIT™ dyestains protein crystals blue, but does not stain salt crystals.

However, in the process of flowing the IZIT™ dye to the crystallizationchamber holding the crystals, the crystals may be dislodged, swept away,and lost. Therefore, the cell pen structure can further be employed incrystallization structures and methods in accordance with the presentinvention to secure crystals in place during the staining process.

FIG. 49 shows an embodiment of a sorting device for crystals based uponthe cell cage concept. Specifically, crystals 8501 of varying sizes maybe formed in flow channel 8502 upstream of sorting device 8500. Sortingdevice 8500 comprises successive rows 8504 of pillars 8506 spaced atdifferent distances. Inlets 8508 of branch channels 8510 are positionedin front of rows 8504. As crystals 8501 flow down channel 8502, theyencounter rows 8504 of pillars 8506. The largest crystals are unable topass between gap Y between pillars 8506 of first row 8504 a, andaccumulate in front of row 8504 a. Smaller sized crystals are gatheredin front of successive rows having successively smaller spacings betweenpillars. Once sorted in the manner described above, the crystals ofvarious sizes can be collected in chambers 8512 by pumping fluid throughbranch channels 8510 utilizing peristaltic pumps 8514 as previouslydescribed. Larger crystals collected by the sorting structure may besubjected to x-ray crystallographic analysis. Smaller crystals collectedby the sorting structure may be utilized as seed crystals in furthercrystallization attempts.

Another factor influencing crystal growth is seeding. Introduction of aseed crystal to the target solution can greatly enhance crystalformation by providing a template to which molecules in solution canalign. Where no seed crystal is available, embodiments of microfluidiccrystallization methods and systems in accordance with the presentinvention may utilize other structures to perform a similar function.

For example, as discussed above, flow channels and chambers ofstructures in accordance with the present invention are typicallydefined by placing an elastomeric layer containing microfabricatedfeatures into contact with an underlying substrate such as glass. Thissubstrate need not be planar, but rather may include projections orrecesses of a size and/or shape calculated to induce crystal formation.In accordance with one embodiment of the present invention, theunderlying substrate could be a mineral matrix exhibiting a regulardesired morphology. Alternatively, the underlying substrate could bepatterned (i.e. by conventional semiconductor lithography techniques) toexhibit a desired morphology or a spectrum of morphologies calculated toinduce crystal formation. The optimal form of such a substrate surfacemorphology could be determined by prior knowledge of the targetcrystals.

Embodiments of crystallization structures and methods in accordance withthe present invention offer a number of advantages over conventionalapproaches. One advantage is that the extremely small volumes(nanoliter/sub-nanoliter) of sample and crystallizing agent permit awide variety of recrystallization conditions to be employed utilizing arelatively small amount of sample.

Another advantage of crystallization structures and methods inaccordance with embodiments of the present invention is that the smallsize of the crystallization chambers allows crystallization attemptsunder hundreds or even thousands of different sets of conditions to beperformed simultaneously. The small volumes of sample and crystallizingagent employed in recrystallization also result in a minimum waste ofvaluable purified target material.

A further advantage of crystallization in accordance with embodiments ofthe present invention is relative simplicity of operation. Specifically,control over flow utilizing parallel actuation requires the presence ofonly a few control lines, with the introduction of sample andcrystallizing agent automatically performed by operation of themicrofabricated device permits very rapid preparation times for a largenumber of samples with the added advantages of parsimonious use ofsample solutions, ease of set-up, creation of well defined fluidicinterfaces, control over equilibration dynamics, and the ability toconduct high-throughput parallel experimentation. These advantages aremade possible by a number of features of the instant invention.

Microfluidics enables the handling of fluids on the sub-nanoliter scale.Consequently, there is no need to use large containment chambers, andhence, assays may be performed on the nanoliter, or subnanoliter scale.The utilization of extremely small volumes allows for thousands ofassays to be performed to consume the same sample volume required forone macroscopic free-interface diffusion experiment. This reduces costlyand time-consuming amplification and purification steps, and makespossible the screening of proteins that are not easily expressed, andhence must be purified from a bulk sample.

Microfluidics further offers savings in preparation times, as hundreds,or even thousands of assays may be performed simultaneously. The use ofscaleable metering techniques as previously described, allow forparallel experimentation to be conducted without increased complexity incontrol mechanisms.

Still another advantage of crystallization systems in accordance withembodiments of the present invention is the ability to control solutionequilibration rates. Crystal growth is often very slow, and no crystalswill be formed if the solution rapidly passes through an optimalconcentration on the way to equilibrium. It may therefore beadvantageous to control the rate of equilibration and thereby promotecrystal growth at intermediate concentrations. In conventionalapproaches to crystallization, slow-paced equilibrium is achieved usingsuch techniques as vapor diffusion, slow dialysis, and very smallphysical interfaces.

However, crystallization in accordance with embodiments of the presentinvention allows for control over the rate of solution equilibrium. Insystems metering crystallizing agent by volume exclusion, the overlyingmembrane can be repeatedly deformed, with each deformation giving riseto the introduction of additional crystallizing agent. In systems thatmeter crystallizing agent by volume entrapment, the valves separatingsample from crystallizing agent may be opened for a short time to allowfor partial diffusive mixing, and then closed to allow chamberequilibration at an intermediate concentration. The process is repeateduntil the final concentration is reached. Either the volume exclusion orentrapment approaches enables a whole range of intermediateconcentrations to be screened in one experiment utilizing a singlereaction chamber. As discussed in detail below, control over kinetics ofthe crystallization process may be controlled by varying the length orcross-sectional area of a capillary connection between reservoirscontaining the sample and crystallizing agent, respectively.

The manipulation of solution equilibrium over time also exploitsdifferential rates of diffusion of macromolecules such as proteinsversus much smaller crystallizing agents such as salts. As large proteinmolecules diffuse much more slowly than the salts, rapidly opening andclosing interface valves allows the concentration of crystallizing agentto be significantly changed, while at the same time very little sampleis lost by diffusion into the larger volume of crystallizing agent.Moreover, as described above, many crystallization structures describedreadily allow for introduction of different crystallizing agents atdifferent times to the same reaction chamber. This allows forcrystallization protocols prescribing changed solvent conditions overtime. Temperature control over equilibration is discussed in detailbelow.

3. Analysis of Crystal Structure from Protein on Chip

The utility of the chip is ultimately dependent on its' ability toquickly generate high quality diffraction patterns at a reduced cost. Aclear path from the chip-to-protein structure is therefore invaluable.Several paths from in-chip crystals to diffraction data are discussedbelow.

One possible application for a chip is determination of favorablecrystallization conditions that can subsequently be reproduced usingconventional techniques. Correspondence between the chip and twoconventional techniques (micro batch and hanging drop) has been shown tobe variable (between 45% and 80%). However, this variation is not afeature unique to the chip. These widely used crystallization techniquesshow only marginal correspondence (e.g. 14 of 16 hanging drop hits forlysozyme do not occur in microbatch), and often show variation withinthemselves. As a tool for screening initial crystallization conditions,the chip may be able to identify as many promising conditions.

FIG. 52 shows a comparison of the number of hits generated on sixdifferent protein samples (lysozyme, glucose isomerase, proteinase K, Bsubunit of topoisomerase VI, xylanase, and bovine pancrease trypsin)using the three different technologies. In FIG. 52 only crystals,microcrystals, rods, and needles are counted as hits, while spherulitesand precipitation is not counted. The data on Proteinase K is a sum ofthe experiments with and without PMSF, and data for the B subunit oftopoisomerase VI has not been included for lack of hanging drop data(although the chip far outperformed micro-batch in this case).Inspection of FIG. 52 shows that in four of the six cases, the chipproduced more hits than either conventional method.

In order to understand differences between crystallization methods toidentify possible reasons for productivity of the chip, we mustappreciate that the three methods produce different thermodynamicconditions on both short and long time scales. In order to induceprotein crystallization, one must make the crystallization energeticallyfavorable (supersaturation condition), and maintain these conditionslong enough for crystal growth to occur.

There are also different degrees of supersaturation. In lowsupersaturation, crystal growth tends to be supported, while nucleationof new crystals is relatively unlikely. In high supersaturation,nucleation is rapid, and many small low quality crystals may often beformed. In the three methods considered here, the condition ofsupersaturation is achieved through the manipulation of the relative,and absolute, concentration of protein and counter-solvent.

A comparison of the phase space evolution/equilibration of the threemethods is shown in FIG. 57. For the micro batch technique, mixing ofthe two solutions is quick, and when kept under impermeable oil layers,little significant concentration occurs over time. Micro batch thereforetends to sample only a single point in phase space, maintainingapproximately the same condition over time.

Hanging drop starts out like micro batch, with rapid mixing of the twosolutions, but then undergoes a concentration on a longer timescale(hours to days) due to vapor equilibration with the more concentratedsalt/precipitant reservoir. During the evaporative dehydration of thedrop, the ratio of protein to precipitant remains constant.

As described in detail below in the description of Microfluidic FreeInterface Diffusion, on the short time scale the chip dynamics mostclosely resemble a free interface diffusion experiment. Mixing is slow,and the rate of species equilibration (protein/precipitant/proton/salt)depends on species' diffusion constants. Small molecules such as saltshave large diffusion constants, and hence equilibrate quickly. Largemolecules (e.g. proteins) have small diffusion constants, andequilibrate more slowly.

The crystallization technique of free interface diffusion in capillariesmay more closely emulate the chip results. Traditionally this method isnot often used due to the difficulty of reliably setting up awell-defined interface. However, in microfluidic environments it isrelatively easy to establish reliable free-interface diffusionexperiments. Additional discussion of the formation of microfluidicfree-interfaces is presented below. In another application of thecrystallization chip, crystals may be grown for harvesting usingconventional methods.

If high quality crystals can be grown in, and extracted from the chip,crystallization conditions need not be exported. Since the chip can beremoved from the glass substrate, it is also possible to extract proteincrystals.

4. Temporal Control Over Equilibration

The growth and quality of crystals is determined not only bythermodynamic conditions explored during the equilibration, but also bythe rate at which equilibration takes place. It is therefore potentiallyvaluable to control the dynamics of equilibration.

In conventional crystallization methods, course control only over thedynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarsely manner. Moreover, once the experiment has begun,no further control over the equilibration dynamics is available.

By contrast, the fluidic interface in a gated μ-Fib experiment may becontrolled by manipulation of the dimensions of the reaction chambersand of the connecting channels. To good approximation, the time requiredfor equilibration varies as the required diffusion length. Theequilibration rate also depends on the cross-sectional area of theconnecting channels. The required time for equilibration may thereforebe controlled by changing both the length, and the cross-sectional areaof the connecting channels.

FIG. 38A shows three sets of pairs of compound chambers 9800, 9802, and9804, each pair connected by microchannels 9806 of a different lengthΔx. FIG. 38B plots equilibration time versus equilibration distance.FIG. 38B shows that the required time for equilibration of the chambersof FIG. 38A varies as the length of the connecting channels.

FIG. 39 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Varying the equilibration rate by changing the geometry of connectingchannels may be used on a single device to explore the effect ofequilibration dynamics on crystal growth. FIGS. 37A-D show an embodimentin which a gradient of concentrations, initially established by thepartial diffusive equilibration of two solutions from a micro-freeinterface, can be maintained by the actuation of containment valves.

FIG. 37A shows flow channel 10000 that is overlapped at intervals by aforked control channel 10002 to define a plurality of chambers (A-G)positioned on either side of a separately-actuated interface valve10004. FIG. 37B plots solvent concentration at an initial time, wheninterface valve 10004 is actuated and a first half 10000 a of the flowchannel has been mixed with a first solution, and a second half 10000 bof the flow channel has been primed with a second solution. FIG. 37Cplots solvent concentration at a subsequent time T₁, when controlchannel 10002 is actuated to define the seven chambers (A-G), whichcapture the concentration gradient at that particular point in time.FIG. 37D plots relative concentration of the chambers (A-G) at time T₁.

In the embodiment shown in FIG. 37A, actuation of the forked controlchannel simultaneously creates the plurality of chambers A-G. However,this is not required, and in alternative embodiments of the presentinvention multiple control channels could be utilized to allowindependent creation of chambers A-G at different time intervals,thereby allow additional diffusion to occur after an initial set ofchambers are created immediately adjacent to the free interface.

An embodiment of a method of capturing a concentration gradient betweentwo fluids comprises providing a first fluid on a first side of anelastomer membrane present within a microfluidic flow channel, andproviding a second fluid on a second side of the elastomer membrane. Theelastomer membrane is displaced from the microfluidic flow channel todefine a microfluidic free interface between the first fluid and thesecond fluid. The first fluid and the second fluid are allowed todiffuse across the microfluidic free interface. A group of elastomervalves positioned along the flow channel at increasing distances fromthe microfluidic free interface are actuated to define a succession ofchambers whose relative concentration of the first fluid and the secondfluid reflects a time of diffusion across the microfluidic freeinterface.

5. Target Materials

Typical targets for crystallization are diverse. A target forcrystallization may include but is not limited to: 1) biologicalmacromolecules (cytosolic proteins, extracellular proteins, membraneproteins, DNA, RNA, and complex combinations thereof), 2) pre- andpost-translationally modified biological molecules (including but notlimited to, phosphorylated, sulfolated, glycosylated, ubiquitinated,etc. proteins, as well as halogenated, abasic, alkylated, etc. nucleicacids); 3) deliberately derivatized macromolecules, such as heavy-atomlabeled DNAs, RNAs, and proteins (and complexes thereof),selenomethionine-labeled proteins and nucleic acids (and complexesthereof), halogenated DNAs, RNAs, and proteins (and complexes thereof),4) whole viruses or large cellular particles (such as the ribosome,replisome, spliceosome, tubulin filaments, actin filaments, chromosomes,etc.), 5) small-molecule compounds such as drugs, lead compounds,ligands, salts, and organic or metallo-organic compounds, and 6)small-molecule/biological macromolecule complexes (e.g., drug/proteincomplexes, enzyme/substrate complexes, enzyme/product complexes,enzyme/regulator complexes, enzyme/inhibitor complexes, and combinationsthereof). Such targets are the focus of study for a wide range ofscientific disciplines encompassing biology, biochemistry, materialsciences, pharmaceutics, chemistry, and physics.

A nonexclusive listing of possible protein modifications is as follows:5′ dephospho; Desmosine (from Lysine); decomposed carboxymethylatedMethionine; Ornithine (from Arginine); Lysinoalanine (from Cysteine);Lanthionine (from Cysteine); Dehydroalanine (from Cysteine); Homoserineformed from Met by CNBr treatment; Dehydration (—H2O); S-gamma-Glutamyl(crosslinked to Cysteine); O-gamma-Glutamyl-(Crosslink to Serine);Serine to Dehydroalanine; Alaminohistidine (Serine crosslinked to thetaor pi carbon of Histidine); Pyroglutamic Acid formed from Gln;N-pyrrolidone carboxyl (N terminus); N alpha-(gamma-Glutamyl)-lysine;N-(beta-Aspartyl)-Lysine (Crosslink); 3,3′,5,5′-TerTyr (Crosslink);Disulphide bond formation (Cystine); S-(2-Histidyl)-(Crosslinked toCysteine); S-(3-Tyr) (Crosslinked to Cysteine); 3,3′-BiTyr (Crosslink);IsodiTyr (Crosslink); Allysine (from Lysine); Amide formation (Cterminus); Deamidation of Asparagine and Glutamine to Aspartate andGlutamate; Citruline (from Arginine); Syndesine (from Lysine);Methylation (N terminus, N epsilon of Lysine, O of Serine, Threonine orC terminus, N of Asparagine); delta-Hydroxy-allysine (from Lysine);Hydroxylation (of delta C of Lysine, beta C of Tryptophan, C3 or C4 ofProline, beta C of Aspartate); Oxidation of Methionine (to Sulphoxide);Sulfenic Acid (from Cysteine); Pyruvoyl-(Serine);3,4-Dihydroxy-Phenylalanine (from Tyrosine) (DOPA); Sodium; Ethyl; N,Ndimethylation (of Arginine or Lysinc); 2,4-BisTrp-6,7-dionc (fromTryptophan); Formylation (CHO); 6,7 Dione (from Tryptophan);3,4,6-Trihydroxy-Phenylalanine (from Tyrosine) (TOPA);3,4-Dihydroxylation (of Proline); Oxidation of Methionine (to Sulphone);3-Chlorination (of Tyrosine with 35Cl); 3-Chlorination (of Tyrosine with37Cl); Potassium; Carbamylation; Acetylation (N terminus, N epsilon ofLysine, O of Serine) (Ac); N-Trimethylation (of Lysine); gammaCarboxylation of Glutamate or beta Carboxylation of Aspartate; disodium;Nitro (NO2); t-butyl ester(OtBu) and t-butyl (tBu); Glycyl (-G-,-Gly-);Carboxymethyl (on Cystine); sodium+potassium; Selenocysteine (fromSerine); 3,5-Dichlorination (of Tyrosine with 35Cl); Dehydroalanine(Dha); 3,5-Dichlorination (of Tyrosine with mixture of 35Cl and 37Cl));Pyruvate; Acrylamidyl or Acrylamide adduct; Sarcosyl; Alanyl (-A-,-Ala-); Acetamidomethyl (Acm); 3,5-Dichlorination (of Tyrosine with37Cl); S-(sn-1-Glyceryl) (on Cysteine); Glycerol Ester (on Glutamic acidside chain); Glycine (G, Gly); Beta mercaptoethanol adduct; Phenyl ester(OPh) (on acidic); 3-Bromination (of Tyrosine with 79Br);Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, Nepsilon of Lysine); 3-Bromination (of Tyrosine with 81Br); Sulphonation(SO3H) (of PMC group); Sulphation (of O of Tyrosine); Cyclohexyl ester(OcHex); Homoseryl lactone; Dehydroamino butyric acid (Dhb); GammaAminobutyryl; 2-Aminobutyric acid (Abu); 2-Aminoisobutyric acid (Aib);Diaminopropionyl; t-butyloxymethyl (Bum); N-(4-NH2-2-OH-butyl)-(ofLysine) (Hypusine); Seryl (-S-, -Ser-); t-butylsulfenyl (StBu); Alanine(A, Ala); Sarcosine (Sar); Anisyl; Benzyl (Bzl) and benzyl ester (OBzl);1,2-ethanedithiol (EDT); Dehydroprolyl; Trifluoroacetyl (TFA);N-hydroxysuccinimide (ONSu, OSu); Prolyl (-P-, -Pro-); Valyl (-V-,-Val-); Isovalyl (-1-,-Iva-); t-Butyloxycarbonyl (tBoc); Threoyl (-T-,-Thr-); Homoseryl (-Hse-); Cystyl (9C9, -Cys-); Benzoyl (Bz);4-Methylbenzyl (Meb); Serine (S, Ser); HMP (hydroxymethylphenyl) linker;Thioanisyl; Thiocresyl; Diphthamide (from Histidine); Pyroglutamyl;2-Piperidinecarboxylic acid (Pip); Hydroxyprolyl (-Hyp-); Norleucyl(-Nle-); Isoleucyl (-I-, -Ile-); Leucyl (-L-, -Leu-); Ornithyl (-Orn-);Asparagyl (-N-, -Asn-); t-amyloxycarbonyl (Aoc); Proline (P, Pro);Aspartyl (-D-, -Asp-); Succinyl; Valine (V, Val); Hydroxybenzotriazoleester (HOBt); Dimethylbenzyl (diMeBzl); Threonine (T, Thr);Cysteinylation; Benzyloxymethyl (Born); p-methoxybenzyl (Mob, Mbzl);4-Nitrophenyl, p-Nitrophenyl (ONp); Cysteine (C, Cys); Chlorobenzyl(ClBzl); Iodination (of Histidine[C4] or Tyrosine[C3]); Glutamyl (-Q-,-Gln-); N-methyl Lysyl; Lysyl (-K-, -Lys-); O-Methyl Aspartamyl;Glutamyl (-E-, -Glu-); N alpha-(gamma-Glutamyl)-Glu; Norleucine (Nle);Hydroxy Aspartamyl; Hydroxyproline (Hyp); bb-dimethyl Cystenyl;Isoleucine (I, Ile); Leucine (L, Leu); Methionyl (-M-, -Met-);Asparagine (N, Asn); Pentoses (Ara, Rib, Xyl); Aspartic Acid (D, Asp);Dmob (Dimethoxybenzyl); Benzyloxycarbonyl (Z); Adamantyl (Ada);p-Nitrobenzyl ester (ONb); Histidyl (-H-, -His-); N-methyl Glutamyl;O-methyl Glutamyl; Hydroxy Lysyl (-Hyl-); Methyl Methionyl; Glutamine(Q, Gln); Aminoethyl Cystenyl; Pentosyl; Deoxyhexoses (Fuc, Rha); Lysine(K, Lys); Aminoethyl cystenyl (-AECys-); 4-Glycosyloxy-(pentosyl, C5)(of Proline); Methionyl Sulfoxide; Glutamic Acid (E, Glu);Phenylalanyl-(-F-, -Phe-); Pyridyl Alanyl; Fluorophenylalanyl;2-Nitrobenzoyl (NBz); Methionine (M, Met); 3-methyl Histidyl;2-Nitrophenylsulphenyl (Nps); 4-Toluenesulphonyl (Tosyl, Tos);3-nitro-2-pyridinesulfenyl (Npys); Histidine (H, His); 3,5-Dibromination(of Tyrosine with 79Br); Arginyl (-R-, -Arg-); Citrulline;3,5-Dibromination (of Tyrosine with mixture of 79Br and 81Br);Dichlorobenzyl (Dcb); 3,5-Dibromination (of Tyrosine with 81 Br);Carboxyamidomethyl Cystenyl; Carboxymethyl Cystenyl; Methylphenylalanyl;Hexosamines (GalN, GlcN); Carboxymethyl cysteine (Cmc); N-Glucosyl (Nterminus or N epsilon of Lysine) (Aminoketose); O-Glycosyl-(to Serine orThreonine); Hexoses (Fm, Gal, Glc, Man); Inositol; MethionylSulphone;Tyrosinyl (-Y-, -Tyr-); Phenylalanine (F, Phe); 2,4-dinitrophenyl (Dnp);Pentafluorophenyl (Pfp); Diphenylmethyl (Dpm); Phospho Seryl;2-Chlorobenzyloxycarbonyl (ClZ); Napthyl acetyl; Isopropyl Lysyl;N-methyl Arginyl; Ethaneditohiol/TFA cyclic adduct; Carboxy Glutamyl(Gla); Acetamidomethyl Cystenyl; Acrylamidyl Cystenyl; Arginine (R,Arg); N-Glucuronyl (N terminus); delta-Glycosyloxy-(of Lysine) orbeta-Glycosyloxy-(of Phenylalanine or Tyrosine); 4-Glycosyloxy-(hexosyl,C6) (of Proline); Benzyl Seryl; N-methyl Tyrosinyl;p-Nitrobenzyloxycarbonyl (4Nz); 2,4,5-Trichlorophenyl;2,4,6-trimethyloxybenzyl (Tmob); Xanthyl (Xan); Phospho Threonyl;Tyrosine (Y, Tyr); Chlorophenylalanyl; Mesitylene-2-sulfonyl (Mts);Carboxymethyl Lysyl; Tryptophanyl (-W-, -Trp-); N-Lipoyl-(on Lysine);Matrix alpha cyano MH+; Benzyl Threonyl; Benzyl Cystenyl; NapthylAlanyl; Succinyl Aspartamyl; Succinimidophenyl carb.; HMP(hydroxymethylphenyl)/TFA adduct; N-acetylhexosamines (GaLNAc, GlcNAc);Tryptophan (W, Trp); Cystine ((Cys)2); Farnesylation; S-Farnesyl-;Myristoleylation (myristoyl with one double bond); PyridylethylCystenyl; Myristoylation; 4-Methoxy-2,3,6-trimethylbenzenesulfonyl(Mtr); 2-Bromobenzyloxycarbonyl (BrZ); Formyl Tryptophanyl; BenzylGlutamyl; Anisole Adducted Glutamyl; S-cystenyl Cystenyl;9-Flourenylmethyloxycarbonyl (Fmoc); Lipoic acid (amide bond to lysine);Biotinylation (amide bond to lysine); Dimethoxybenzhydryl (Mbh);N-Pyridoxyl (on Lysine); Pyridoxal phosphate (Schiff Base formed tolysine); Nicotinyl Lysyl; Dansyl (Dns);2-(p-biphenyl)isopropyl-oxycarbonyl (Bpoc); Palmitoylation;“Triphenylmethyl (Trityl, Trt)”; Tyrosinyl Sulphate; Phospho Tyrosinyl;Pbf (pentamethyldihydrobenzofuransulfonyl); 3,5-Diiodination (ofTyrosine); 3,5-di-I″; N alpha-(gamma-Glutamyl)-Glu2;O-GlcNAc-1-phosphorylation (of Serine);“2,2,5,7,8-Pentamethylchroman-6-sulphonyl (Pmc)”; Stearoylation;Geranylgeranylation; S-Geranylgeranyl; 5′phos dCytidinyl; iodoTyrosinyl; Aldohexosyl Lysyl; Sialyl; N-acetylneuraminic acid (Sialicacid, NeuAc, NANA, SA); 5′phos dThymidinyl; 5′phos Cytidinyl;Glutathionation; O-Uridinylylation (of Tyrosine); 5′phos Uridinyl;S-farnesyl Cystenyl; N-glycolneuraminic acid (NeuGc); 5′phos dAdenosyl;O-pantetheinephosphorylation (of Serine); SucPhencarb Lysyl; 5′phosdGuanosyl; 5′phos Adenosinyl; O-5′-Adenosylation (of Tyrosine);4′-Phosphopantetheine; GL2; S-palmityl Cystenyl; 5′phos Guanosyl;Biotinyl Lysyl; Hex-HexNAc; N alpha-(gamma-Glutamyl)-Glu3; DioctylPhthalate; PMC Lysyl; Aedans Cystenyl; Dioctyl Phthalate Sodium Adduct;di-iodo Tyrosinyl; PMC Arginyl; S-Coenzyme A; AMP Lysyl;3,5,3′-Triiodothyronine (from Tyrosine);S-(sn-1-Dipalmitoyl-glyceryl)-(on Cysteine); S-(ADP-ribosyl)-(onCysteine); N-(ADP-ribosyl)-(on Arginine); O-ADP-ribosylation (onGlutamate or C terminus); ADP-rybosylation (from NAD); S-Phycocyanobilin(on Cysteine); S-Heme (on Cysteine); N theta-(ADP-ribosyl) diphthamide(of Histidine); NeuAc-Hex-HexNAc; MGDG; O-8 alpha-Flavin [FAD])-(ofTyrosine); S-(6-Flavin [FAD])-(on Cysteine); N theta and Npi-(8alpha-Flavin) (on Histidine); (Hex)3-HexNAc-HexNAc;(Hex)3-HexNAc-(dHex)HexNAc.

A nonexclusive listing of possible nucleic acid modifications, such asbase-specific, sugar-specific, or phospho-specific is as follows:halogenation (F, Cl, Br, I); Abasic sites; Alkylation; Crosslinkableadducts such as thiols or azides; Thiolation; Deamidation;Fluorescent-group labeling, and glycosylation.

A nonexclusive listing of possible heavy atom derivatizing agents is asfollows: potassium hexachloroiridate (III); Potassium hexachloroiridate(IV); Sodium hexachloroiridate (IV); Sodium hexachloroiridate (III);Potassium hexanitritoiridate (III); Ammnoium hexachloroiridate (III);Iridium (III) chloride; Potassium hexanitratoiridate (III); Iridium(III) bromide; Barium (II) chloride; Barium (II) acetate; Cadmium (II)nitrate; Cadmium (II) iodide; Lead (II) nitrate; Lead (II) acetate;Trimethyl lead (IV) chloride; Trimethyl lead (IV) acetate; Ammoniumhexachloro plumbate (IV); Lead (II) chloride; Sodium hexachlororhodiate(III); Strontium (II) acetate; Disodium thiomalonato aurate (I);Potassium dicyano aurate (I); Sodium dicyano aurate (I); Sodiumthiosulphato aurate (III); Potassium tetracyano aurate (III); Potassiumtetrachloro aurate (III); Hydrogen tetrachloro aurate (III); Sodiumtetrachloro aurate (III); Potassium tetraiodo aurate (III); Potassiumtetrabromo aurate (III); (acetato-o)methylmercury; Methyl (nitrato-o)mercury; Chloromethylmercury; Iodomethylmercury; Chloroethylmercury;Methyl mercury cation; Triethyl (m3-phosphato(3-)-0,0′,0″)tri mercuryeth; [3-[(aminocarbonyl)amino]-2-methoxypropyl]chlorome; 1,4diacetoxymercury 2-3 dimethoxy butane; Meroxyl mercuhydrin; Tetrakis(acetoxy mercuri)-methane; 1,4-bis(chloromercuri)-2,3-butanediol; Ethyldiacetoxymercurichloro acetate (dame); Mercuric (II) oxide; Methylmercuri-2-mercaptoethanol; 3,6 bis(mercurimethyl dioxane acetate); Ethylmercury cation; Billman's dimercurial; Para chloromercury phenyl acetate(pcma); Mercury phenyl glyoxal (mpg); Thiomersal, ethyl mercurythiosalicylate [emts]; 4-chloromercuribenesulphonic acid; 2,6dichloromercuri-4-nitrophenol (dcmnp); [3-[[2(carboxymethoxy)benzoyl]mino-2 methoxy prop; Parachloromercury benzoate (pcmb), 4-chloromercury;(acetato-o)phenyl mercury; Phenyl mercuri benzoate (pmb); Para hydroxymercuri benzoate (phmb); Mercuric imidosuccinate/mercury succinimide;3-hydroxymercurybenzaldehyde; 2-acetoxy mercuri sulhpanilamide;3-acetoxymercuri-4-aminobenzenesulphonamide; Methyl mercuri thioclycolicacid (mmtga); 2-hydroxymercuri-tolulen-4-sulphonic acid (hmts);Acetamino phenyl mercury acetate (apma);[3-[(aminocarbonyl)amino]-3-methoxypropyl 2-chloro; Para-hydroxymercuribenzene sulphonate (phmbs); Ortho-chloromercuri phenol (ocmp);Diacetoxymercury dipopylene dioxide (dmdx); Para-acetoxymercuri aniline(pama); (4-aminophenyl) chloromercury; Aniline mercury cation;3-hydroxy-mercuri-s sulphosalicylic acid (msss); 3 or 5 hydroxymercurisalicylic acid (hmsa); Diphenyl mercury; 2,6 diacetoxymercurimethyl 1-4thioxane (dmmt); 2,5-b1s (chloromercury) furan; Ortho-chloromercurinitrophenol (ocmnp); 5-mercurydeoxyuridine monosulphate; Mercurysalicylate; [3-[[2-(carboxymethoxy)benzoyl]amino-2-methoxypro; 3,3bis(hydroximercuri)-3-nitratomercuri pyruvic; 3-chloro mercuri pyridine;3,5 bis acetoxymercuri methyl morpholine; Ortho-mercury phenol cation;Para-carboxymethyl mercaptomercuri benzensulphonyl; Para-mercuribenzoylglucosamine; 3-acetetoxyrnercuri-5-nitrosalicyladehyde (msa); Ammoniumtetrachloro mercurate (II); Potassium tetrathiocyanato mercurate (II);Sodium tetrathiocyanato mercurate (II); Potassium tetraisothiocyantomercurate (II); Potassium tetraido mercurate (II); Ammoniumtetrathiocyananato mercurate (II); Potassium tetrabromo mercurate (II);Potassium tetracyano mercurate (II); Mercury (II) bromide; Mercury (II)thiocyanate; Mercury (II) cyanide; Mercury (II) iodide; Mercuric (II)chloride; Mercury (II) acetate; Mercury (I) acetate; Dichlorodiaminomercurate (II); Beta mercury-mercapto-ethylamine hydrochloride; Mercury(II) sulphate; Mercury (II) chloroanilate; Dimercuriacetate;Chloro(2-oxoethyl) mercury; Phenol mercury nitrate; Mercurymercaptoethanol; Mercury mercaptoethylamine chloride; Mercurythioglycollic acid (sodium salt);0-hydroxymercuri-p-nitrophenol/2-hydroxymercuri-4-; Para chloromercuriphenol (pcmp); Acetylmercurithiosalicylate (amts); Iodine; Potassiumiodide (iodine); 4-iodopyrazole; O-iodobenzoylglucasamine;P-iodobenzoylglucasamine; Potassium iodide/chloramine t; Ammoniumiodide; 3-isothio-cyanato-4-iodobenzene sulphonate; Potassium iodide;3′-iodo phenyltrazine; 4′-iodo phenyltrazine; Sodium iodide/iodine;Silver nitrate; Silver ( ) trinitridosulphoxylate; Tobenamed; Samarium(III) chloride; Thulium (III) chloride; Lutetium (III) chloride;Europium (III) chloride; Terbium (III) chloride; Gadolinium (III)chloride; Erbium (III) chloride; Lanthanum (III) chloride; Samarium(III) nitrate; Samarium (III) acetate; Samarium (III) cation;Praseodymium (III) chloride; Neodymium (III) chloride; Ytterbium (III)chloride; Thulium (III) sulphate; Ytterbium (III) sulphate; Gadolinium(III) sulphate; Gadolinium (III) acetate; Dysprosium (III) chloride;Erbium (III) nitrate; Holmium (III) chloride; Penta amino ruthenium(III) chloride; Cesium nitridotiroxo osmium (viii); Potassium tetraoxoosmiate; Hexa amino osmium (III) iodide; Ammonium hexachloroosmiate(IV); Osmium (III) chloride; Potassium hexachloro osmiate (IV); Cesiumtrichloro triscarbonyl osmiate (?); Dinitritodiamine platinum (II); Cisdichlorodimethylammido platinum (II); Dichlorodiammine platinum (II);Dibromodiammine platinum (II); Dichloroethylene diamine platinum (II);Potassium dicholodinitrito platinate (II); Diethylenediamene platinum(II); Potassium dioxylato platinate (II); Dichlorobis (pyridine)platinum (II); Potassium (thimethyl dibenzyloamine) platinum; Potassiumtetrabromoplatinate (II); Potassium tetrachloro platinate (II);Potassium tetranitrito platinum (II); Potassium tetracyano platinate(II); Sodium tetracyano platinate (II); Potassium tetrathiocyanatoplatinate (II); Ammonium tetranitrito platinate (II); Potassiumtetraisocyanato platinate (II); Ammonium tetracyano platinum (II);Ammonium tetrachloro platinate (II); Potassium dinitritodioxalatoplatinate (IV); Dichlorotetraammino platinium (IV); Dibromodinitritodiammine platinium (IV); Potassium hexanitrito platinate (IV); Potassiumhexachloro platinate (IV); Potassium hexabromo platinate (IV); Sodiumhexachloroplatinate (IV); Potassium hexaiodo platinate (IV); Potassiumhexathiocyanato platinate (IV); Tetrachloro bis(pyridine) platinum (IV);Ammonium hexachloro platinate (IV); Di-mu-iodo bis(ethylenediamine) diplatinum (II) n; Potassium hexaisothiocyanato platinate (IV); Potassiumtetraiodo platinate (II); 2,2′,2″ terpyridyl platinium (II); 2hydroxyethanethiolate (2,2′,2″ terpyeidine) pla; Potassium tetranitroplatinate (II); Trimethyl platinum (II) nitrate; Sodium tetraoxo rhenate(VII); Potassium tetraoxo rhenate (VI); Potassium tetraoxo rhenate(VII); Potassium hexachloro rhenium (IV); Rhenium (III) chloride;Ammonium hexachloro rhenate (IV); Dimethyltin (II) dichloride; Thorium(IV) nitrate; Uranium (VI) oxychloride; Uranium (VI) oxynitrate; Uranium(VI) oxyacetate; Uranium (VI) oxypyrophosphate; Potassium pentafluorooxyuranate (VI); Sodium pentafluoro oxyuranate (VI); Potassiumnanofluoro dioxyuranate (VI); Sodium triacetate oxyuranate (VI); Uranium(VI) oxyoxalate; Selenocyanate anion; Sodium tungstate; Sodium12-tungstophosphate; Thallium (I) acetate; Thallium (I) fluoride;Thallium (I) nitrate; Potassium tetrachloro palladate (II); Potassiumtetrabromo palladate (II); Potassium tetracyano palladate (II);Potassium tetraiodo palladate (II); Cobalt (II) chloride.

The PDMS material from which the chip can be formed is well suited formany of these targets, particularly biological samples. PDMS is anon-reactive and biologically inert compound that allows such moleculesto maintain their appropriate shape, fold, and activity in a solublizedstate. The matrix and system can accommodate a range of target sizes andmolecular weights, from a few hundred Daltons to the mega-Dalton regime.Biological targets, from small proteins and peptides to viruses andmacromolecular complexes, fall within this range, and are generallyanywhere from 3-10 kDa to >1-2 MDa in size.

6. Solute/Reagent Types

During crystallization screening, a large number of chemical compoundsmay be employed. These compounds include salts, small and largemolecular weight organic compounds, buffers, ligands, small-moleculeagents, detergents, peptides, crosslinking agents, and derivatizingagents. Together, these chemicals can be used to vary the ionicstrength, pH, solute concentration, and target concentration in thedrop, and can even be used to modify the target. The desiredconcentration of these chemicals to achieve crystallization is variable,and can range from nanomolar to molar concentrations. A typicalcrystallization mix contains set of fixed, but empirically-determined,types and concentrations of ‘precipitants’, buffers, salts, and otherchemical additives (e.g., metal ions, salts, small molecular chemicaladditives, cryo protectants, etc.). Water is a key solvent in manycrystallization trials of biological targets, as many of these moleculesmay require hydration to stay active and folded.

As described above in connection with the pressurized out-gas priming(POP) technique, the permeability of PDMS to gases, and thecompatibility of solvents with PDMS may be a significant factor indeciding upon precipitating agents to be used.

‘Precipitating’ agents act to push targets from a soluble to insolublestate, and may work by volume exclusion, changing the dielectricconstant of the solvent, charge shielding, and molecular crowding.Precipitating agents compatible with the PDMS material of certainembodiments of the chip include, but are not limited to, non-volatilesalts, high molecular weight polymers, polar solvents, aqueoussolutions, high molecular weight alcohols, divalent metals.

Precipitating compounds, which include large and small molecular weightorganics, as well as certain salts are used from under 1% to upwards of40% concentration, or from <0.5M to greater than 4M concentration. Wateritself can act in a precipitating manner for samples that require acertain level of ionic strength to stay soluble. Many precipitants mayalso be mixed with one another to increase the chemical diversity of thecrystallization screen. The microfluidics devices described in thisdocument are readily compatible with a broad range of such compounds.Moreover, many precipitating agents (such as long- and short-chainorganics) are quite viscous at high concentrations, presenting a problemfor most fluid handling devices, such as pipettes or robotic systems.The pump and valve action of microfluidics devices in accordance withembodiments of the present invention enable handling of viscous agents.

An investigation of solvent/precipitating agent compatibility withparticular elastomer materials may be conducted to identify optimumcrystallizing agents, which may be employed develop crystallizationscreening reactions tailored for the chip that are more effective thanstandard screens.

A nonexclusive list of salts which may be used as precipitants is asfollows: Tartrate (Li, Na, K, Na/K, NH4); Phosphate (Li, Na, K, Na/K,NH4); Acetate (Li, Na, K, Na/K, Mg, Ca, Zn, NH4); Formate (Li, Na, K,Na/K, Mg, NH4); Citrate (Li, Na, K, Na/K, NH4); Chloride (Li, Na, K,Na/K, Mg, Ca, Zn, Mn, Cs, Rb, NH4); Sulfate (Li, Na, K, Na/K, NH4);Malate (Li, Na, K, Na/K, NH4); Glutamate (Li, Na, K, Na/K, NH4.

A nonexclusive list of organic materials which may be used asprecipitants is as follows: PEG 400; PEG 1000; PEG 1500; PEG 2k; PEG3350; PEG 4k; PEG 6k; PEG 8k; PEG 10k; PEG 20k; PEG-MME 550; PEG-MME750; PEG-MME 2k; PEG-MME 5k; PEG-DME 2k; Dioxane; Methanol; Ethanol;2-Butanol; n-Butanol; t-Butanol; Jeffamine M-600; Isopropanol;2-methyl-2,4-pentanediol; 1,6 hexanediol.

Solution pH can be varied by the inclusion of buffering agents; typicalpH ranges for biological materials lie anywhere between values of3.5-10.5 and the concentration of buffer, generally lies between 0.01and 0.25 M. The microfluidics devices described in this document arereadily compatible with a broad range of pH values, particularly thosesuited to biological targets.

A nonexclusive list of possible buffers is as follows: Na-Acetate;HEPES; Na-Cacodylate; Na-Citrate; Na-Succinate; Na-K-Phosphate; TRIS;TRIS-Maleate; Imidazole-Maleate; BisTrisPropane; CAPSO, CHAPS, MES, andimidizole.

Additives are small molecules that affect the solubility and/or activitybehavior of the target. Such compounds can speed crystallizationscreening or produce alternate crystal forms of the target. Additivescan take nearly any conceivable form of chemical, but are typically monoand polyvalent salts (inorganic or organic), enzyme ligands (substrates,products, allosteric effectors), chemical crosslinking agents,detergents and/or lipids, heavy metals, organo-metallic compounds, traceamounts of precipitating agents, and small molecular weight organics.

The following is a nonexclusive list of possible additives: 2-Butanol;DMSO; Hexanediol; Ethanol; Methanol; Isopropanol; sodium fluoride;potassium fluoride; ammonium fluoride; lithium chloride anhydrous;magnesium chloride hexahydrate; sodium chloride; Calcium chloridedihydrate; potassium chloride; ammonium chloride; sodium iodide;potassium iodide; ammonium iodide; sodium thiocyanate; potassiumthiocyanate; lithium nitrate; magnesium nitrate hexahydrate; sodiumnitrate; potassium nitrate; ammonium nitrate; magnesium formate; sodiumformate; potassium formate; ammonium formate; lithium acetate dihydrate;magnesium acetate tetrahydrate; zinc acetate dihydrate; sodium acetatetrihydrate; calcium acetate hydrate; potassium acetate; ammoniumacetate; lithium sulfate monohydrate; magnesium sulfate heptahydrate;sodium sulfate decahydrate; potassium sulfate; ammonium sulfate;di-sodium tartate dihydrate; potassium sodium tartrate tetrahydrate;di-ammonium tartrate; sodium dihydrogen phosphate monohydrate; di-sodiumhydrogen phosphate dihydrate; potassium dihydrogen phosphate;di-potassium hydrogen phosphate; ammonium dihydrogen phosphate;di-ammonium hydrogen phosphate; tri-lithium citrate tetrahydrate;tri-sodium citrate dihydrate; tri-potassium citrate monohydrate;di-ammonium hydrogen citrate; barium chloride; cadmium chloridedihydrate; cobaltous chloride dihydrate; cupric chloride dihydrate;strontium chloride hexahydrate; yttrium chloride hexahydrate; ethyleneglycol; Glycerol anhydrous; 1,6 hexanediol; MPD; polyethylene glycol400; trimethylamine HCl; guanidine HCl; urea; 1,2,3-heptanetriol;benzamidine HCl; dioxane; ethanol; iso-propanol; methanol; sodiumiodide; L-cysteine; EDTA sodium salt; NAD; ATP disodium salt;D(+)-glucose monohydrate; D(+)-sucrose; xylitol; spermidine; sperminetetra-HCl; 6-aminocaproic acid; 1,5-diaminopentanc di-HCl;1,6-diaminohexanc; 1,8-diaminooctane; glycine; glycyl-glycyl-glycine;hexaminecobalt trichloride; taurine; betaine monohydrate;polyvinylpyrrolidone K15; non-detergent sulfo-betaine 195; non-detergentsulfo-betaine 201; phenol; DMSO; dextran sulfate sodium salt; jeffamineM-600; 2,5 Hexanediol; (+/+)1,3 butanediol; polypropylene glycol P400;1,4 butanediol; tert-butanol; 1,3 propanediol; acetonitrile; gammabutyrolactone; propanol; ethyl acetate; acetone; dichloromethane;n-butanol; 2,2,2 trifluoroethanol; DTT; TCEP; nonaethylene glycolmonododecyl ether, nonaethylene glycol monolauryl ether; polyoxyethylene(9) ether; octaethylene glycol monododecyl ether, octaethylene glycolmonolauryl ether; polyoxyethylene (8) lauryl ether;Dodecyl-β-D-maltopyranoside; Lauric acid sucrose ester;Cyclohexyl-pentyl-β-D-maltoside; Nonaethylene glycol octylphenol ether;Cetyltrimethylammonium bromide;N,N-bis(3-D-gluconamidopropyl)-deoxycholamine;Decyl-β-D-maltopyranoside; Lauryldimethylamine oxide;Cyclohexyl-pentyl-β-D-maltoside; n-Dodecylsulfobetaine,3-(Dodecyldimethylammonio)propane-1-sulfonate;Nonyl-β-D-glucopyranoside; Octyl-β-D-thioglucopyranoside, OSG;N,N-Dimethyldecylamine-β-oxide;Methyl-6-O-(N-heptylcarbamoyl)-a-D-glucopyranoside; Sucrosemonocaproylate; n-Octanoyl-β-D-fructofuranosyl-a-D-glucopyranoside;Heptyl-β-D-thioglucopyranoside; Octyl-β-D-glucopyranoside, OG;Cyclohexyl-propyl-β-D-maltoside;Cyclohexylbutanoyl-N-hydroxyethylglucamide; n-decylsulfobetaine,3-(Decyldimethylammonio)propane-1-sulfonate; Octanoyl-N-methylglucamide,OMEGA; Hexyl-β-D-glucopyranoside; Brij 35; Brij 58; Triton X-114; TritonX-305; Triton X-405; Tween 20; Tween 80; polyoxyethylene(6)decyl ether;polyoxyethylene(9)decyl ether; polyoxyethylene(10)dodecyl ether;polyoxyethylene(8)tridecyl ether; Isopropyl-β-D-thiogalactoside;Decanoyl-N-hydroxyethylglucamide; Pentaethylene glycol monooctyl ether;3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate;3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propane sulfonate;Cyclohexylpentanoyl-N-hydroxyethylglucamide;Nonanoyl-N-hydroxyethyglucamide;Cyclohexylpropanol-N-hydroxyethylglucamide;Octanoyl-N-hydroxyethylglucamide;Cyclohexylethanoyl-N-hydroxyethylglucamide; Benzyldimethyldodecylammonium bromide; n-Hexadecyl-β-D-maltopyranoside;n-Tetradecyl-β-D-maltopyranoside; n-Tridecyl-β-D-maltopyranoside;Dodecylpoly(ethyleneglycoether)n;n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;n-Undecyl-β-D-maltopyranoside; n-Decyl-β-D-thiomaltopyranoside;n-dodecylphosphocholine; a-D-glucopyranoside, β-D-fructofuranosylmonodecanoate, sucrose mono-caprate; 1-s-Nonyl-β-D-thioglucopyranoside;n-Nonyl-β-D-thiomaltoyranoside; N-Dodecyl-N,N-(dimethlammonio)butyrate;n-Nonyl-β-D-maltopyranoside; Cyclohexyl-butyl-β-D-maltoside;n-Octyl-β-D-thiomaltopyranoside; n-Decylphosphocholine;n-Nonylphosphocholine; Nonanoyl-N-methylglucamide;1-s-Heptyl-β-D-thioglucopyranoside; n-Octylphosphocholine;Cyclohexyl-ethyl-β-D-maltoside;n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;Cyclohexyl-methyl-β-D-maltoside.

Cryosolvents are agents that stabilize a target crystal to flash-coolingin a cryogen such as liquid nitrogen, liquid propane, liquid ethane, orgaseous nitrogen or helium (all at approximately 100-120° K) such thatcrystal becomes embedded in a vitreous glass rather than ice. Any numberof salts or small molecular weight organic compounds can be used as acryoprotectant, and typical ones include but are not limited to: MPD,PEG-400 (as well as both PEG derivatives and higher molecular-weight PEGcompounds), glycerol, sugars (xylitol, sorbitol, erythritol, sucrose,glucose, etc.), ethylene glycol, alcohols (both short- and long chain,both volatile and nonvolatile), LiOAc, LiCl, LiCHO₂, LiNO₃, Li2SO₄,Mg(OAc)₂, NaCl, NaCHO₂, NaNO₃, etc. Again, materials from whichmicrofluidics devices in accordance with the present invention arefabricated may be compatible with a range of such compounds.

Many of these chemicals can be obtained in predefined screening kitsfrom a variety of vendors, including but not limited to Hampton Researchof Laguna Niguel, Calif., Emerald Biostructures of Bainbridge Island,Wash., and Jena BioScience of Jena, Germany, that allow the researcherto perform both ‘sparse matrix’ and ‘grid’ screening experiments. Sparsematrix screens attempt to randomly sample as much of precipitant,buffer, and additive chemical space as possible with as few conditionsas possible. Grid screens typically consist of systematic variations oftwo or three parameters against one another (e.g., precipitantconcentration vs. pH). Both types of screens have been employed withsuccess in crystallization trials, and the majority of chemicals andchemical combinations used in these screens are compatible with the chipdesign and matrices in accordance with embodiments of the presentinvention.

Moreover, current and future designs of microfluidic devices may enableflexibly combinatorial screening of an array of different chemicalsagainst a particular target or set of targets, a process that isdifficult with either robotic or hand screening. This latter aspect isparticularly important for optimizing initial successes generated byfirst-pass screens.

7. Additional Screening Variables For Crystallization

In addition to chemical variability, a host of other parameters can bevaried during crystallization screening. Such parameters include but arenot limited to: 1) volume of crystallization trial, 2) ratio of targetsolution to crystallization solution, 3) target concentration, 4)co-crystallization of the target with a secondary small ormacromolecule, 5) hydration, 6) incubation time, 7) temperature, 8)pressure, 9) contact surfaces, 10) modifications to target molecules,and 11) gravity.

Volumes of crystallization trials can be of any conceivable value, fromthe picoliter to milliliter range. Typical values may include but arenot limited to: 0.1, 0.2, 0.25, 0.4, 0.5, 0.75, 1, 2, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200,225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 900,1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2250, 2500, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, and 10000nL. The microfluidics devices previously described can access thesevalues.

In particular, access to the low volume range for crystallization trials(<100 nL) is a distinct advantage of embodiments of the microfluidicschips in accordance with embodiments of the present invention, as suchsmall-volume crystallization chambers can be readily designed andfabricated, minimizing the need the need for large quantities ofprecious target molecules. The low consumption of target material ofembodiments in accordance with the present invention is particularlyuseful in attempting to crystallize scarce biological samples such asmembrane proteins, protein/protein and protein/nucleic acid complexes,and small-molecule drug screening of lead libraries for binding totargets of interest.

The ratios of a target solution to crystallization mix can alsoconstitute an important variable in crystallization screening andoptimization. These rations can be of any conceivable value, but aretypically in the range of 1:100 to 100:1 target:crystallization-solution. Typical target: crystallization-solution orcrystallization-solution: target ratios may include but are not limitedto: 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:25, 1:20, 1:15,1:10, 1:9, 1:8, 1:7.5, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:3,3:4, 3:5, 4:5, 5:6, 5:7, 5:9, 6:7, 7:8, 8:9, and 9:10. As previouslydescribed, microfluidics devices in accordance with embodiments of thepresent invention can be designed to access multiple ratiossimultaneously on a single chip.

Target concentration, like crystallization chemical concentration, canlie in a range of values and is an important variable in crystallizationscreening. Typical ranges of concentrations can be anywhere from <0.5mg/ml to >100 mg/ml, with most commonly used values between 5-30 mg/ml.The microfluidics devices in accordance with embodiments of the presentinvention are readily compatible with this range of values.

Co-crystallization generally describes the crystallization of a targetwith a secondary factor that is a natural or non-natural bindingpartner. Such secondary factors can be small, on the order of about10-1000 Da, or may be large macromolecules. Co-crystallization moleculescan include but are not limited to small-molecule enzyme ligands(substrates, products, allosteric effectors, etc.), small-molecule drugleads, single-stranded or double-stranded DNAs or RNAs, complementproteins (such as a partner or target protein or subunit), monoclonalantibodies, and fusion-proteins (e.g., maltose binding proteins,glutathione S-transferase, protein-G, or other tags that can aidexpression, solubility, and target behavior). As many of these compoundsare either biological or of a reasonable molecular weight,co-crystallization molecules can be routinely included with screens inthe microfluidics chips. Indeed, because many of these reagents areexpensive and/or of limited quantity, the small-volumes afforded by themicrofluidics chips in accordance with embodiment of the presentinvention make them ideally suited for co-crystallization screening.

Hydration of targets can be an important consideration. In particular,water is by far the dominant solvent for biological targets and samples.The microfluidics devices described in this document are relativelyhydrophobic, and are compatible with water-based solutions.

The length of time for crystallization experiments can range fromminutes or hours to weeks or months. Most experiments on biologicalsystems typically show results within 24 hours to 2 weeks. This regimeof incubation time can be accommodated by the microfluidics devices inaccordance with embodiments of the present invention.

The temperature of a crystallization experiment can have a great impacton success or failure rates. This is particularly true for biologicalsamples, where temperatures of crystallization experiments can rangefrom 0-42° C. Some of the most common crystallization temperatures are:0, 1, 2, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 37, and 42.Microfluidics devices in accordance with embodiments of the presentinvention can be stored at the temperatures listed, or alternatively maybe placed into thermal contact with small temperature control structuressuch as resistive heaters or Peltier cooling structures.

In addition, the small footprint and rapid setup time of embodiments inaccordance with the present invention allow faster equilibration todesired target temperatures and storage in smaller incubators at a rangeof temperatures. Moreover, as the microfluidics systems in accordancewith embodiments of the present invention do not place thecrystallization experiment in contact with the vapor phase, condensationof water from the vapor phase into the drop as temperatures change, aproblem associated with conventional macroscopic vapor-diffusiontechniques, is avoided. This feature represents an advance over manyconventional manual or robotic systems, where either the system must bemaintained at the desired temperature, or the experiment must remain atroom temperature for a period before being transferred to a newtemperature.

Variation in pressure is an as yet understudied crystallizationparameter, in part because conventional vapor-diffusion and microbatchprotocols do not readily allow for screening at anything typically otherthan atmospheric pressure. The rigidity of the PDMS matrix enablesexperiments to probe the effects of pressure on target crystallizationon-chip.

The surface on which the crystallization ‘drop’ sits can affectexperimental success and crystal quality. Examples of solid supportcontact surfaces used in vapor diffusion and microbatch protocolsinclude either polystyrene or silanized glass. Both types of supportscan show different propensities to promote or inhibit crystal growth,depending on the target. In addition, the crystallization ‘drop’ is incontact with either air or some type of poly-carbon oil, depending onwhether the experiment is a vapor-diffusion or microbatch setup,respectively. Air contact has the disadvantage in that free oxygenreacts readily with biological targets, which can lead to proteindenaturation and inhibit or degrade crystallization success. Oil allowstrace hydrocarbons to leach into the crystallization experiment, and cansimilarly inhibit or degrade crystallization success.

Microfluidics device designs in accordance with embodiments of thepresent invention may overcome these limitations by providing anonreactive, biocompatible environment that completely surrounds thecrystallization reaction. Moreover, the composition of thecrystallization chambers in the microfluidics chips can conceivably bevaried to provide new surfaces for contacting the crystallizationreaction; this would allow for routine screening of different surfacesand surface properties to promote crystallization.

Crystallization targets, particularly those of biological origin, mayoften be modified to enable crystallization. Such modifications includebut are not limited to truncations, limited proteolytic digests,site-directed mutants, inhibited or activated states, chemicalmodification or derivatization, etc. Target modifications can be timeconsuming and costly; modified targets require the same thoroughscreening as do unmodified targets. Microfluidics devices of the presentinvention work with such modified targets as readily as with theoriginal target, and provide the same benefits.

The effect of gravity as a parameter for crystallization is yet anotherunderstudied crystallization parameter, because of the difficulty ofvarying such a physical property. Nonetheless, crystallizationexperiments of biological samples in zero gravity environments haveresulted in the growth of crystals of superior quality than thoseobtained on Earth under the influence of gravity.

The absence of gravity presents problems for traditional vapor-diffusionand microbatch setups, because all fluids must be held in place bysurface tension. The need to often set up such experiments by hand alsoposes difficulties because of the expense of maintaining personnel inspace. Microfluidics devices in accordance with embodiments of thepresent invention, however, would enable further exploration ofmicrogravity as a crystallization condition. A compact, automatedmetering and crystal growth system would allow for: 1) launching ofsatellite factory containing target molecules in a cooled, but liquidstate, 2) distribution of targets and growth of crystals, 3) harvestingand cryofreezing of resultant crystals, and 4) return of cryo-storedcrystals to land-based stations for analysis.

8. In Situ Crystallization Screening

The ability to observe the growth of crystals with a microscope is astep in deciding upon success or failure of crystallization trials.Conventional crystallization protocols may use transparent materialssuch as polystyrene or silanized glass to allow for visualization. Thetransparency of the PDMS matrix of embodiments in accordance with thepresent invention is particularly suited to the two primary methods bywhich crystallization trials are traditionally scored: 1) directobservation in the visible light regime by optical microscopes and 2)birefringence of polarized light.

Birefringence may be difficult to judge in conventional experiments asmany plastics are themselves birefringent, interfering with sampleassessment. However, the microfluidics devices described herein can bemade without such optical interference properties, allowing for thedesign of an automated scanning system that routinely allows directvisualization with both polarizing and non-polarizing features.

In addition, robotic and, in particular, manually-set crystallizationexperiments can vary the placement of a crystallization drop on asurface by tens to hundreds of microns. This variability presents aproblem for automated scanning systems, as it is difficult to program inthe need for such flexible positioning without stable fiducials.However, the fixed placement of crystallization chambers in themicrofluidics chips of embodiments of the present invention overcomessuch problems, as every well can be positioned in a particular locationwith submicron accuracy. Moreover, such a system is readily scalable forthe design of differently sized and positioned crystallization chambers,as masks and other templates used to design microfluidics devices inaccordance with embodiments of the present invention can be simplydigitized and ported into scanning software for visualization.

Once crystals are obtained by visual inspection, it may be possible toscreen for diffraction directly through the chip itself. For example, acrystallization chamber within a chip could be outfitted withtransparent ‘windows’ comprising glass, quartz, or thinned portions ofthe elastomer material itself, on opposite walls of the chamber.Crystals could then be exposed directly to x-rays through the chip toassay for diffraction capabilities, eliminating the need to remove, andthereby possibly damage, the crystalline sample. Such an approach couldbe used to screen successes from initial crystallization trials todetermine the best starting candidate conditions for follow-up study.Similarly, crystals grown under a particular set of conditions could be‘re-equilibrated’ with new solutions (e.g., cryo-stabilizing agents,small-molecule drug leads or ligands, etc.), and the stability of thecrystals to such environment changes monitored directly by x-raydiffraction.

9. Utilizing Microfluidic Devices For Purification/Crystallization

Crystallization of target biological samples such as proteins isactually the culmination of a large number of prior complex anddifficult steps, including but not limited to protein expression,purification, derivatization, and labeling. Such steps prior tocrystallization comprise shuttling liquids from a chamber with one setof solution properties to another area with a different set ofproperties. Mircofluidics technology is suited to perform such tasks,allowing for the combination of all necessary steps within the confinesof a single chip.

Examples of microfluidic handling structures enabling performance ofpre-crystallization steps have been described above. For example, amicrofluidics chip could act as a regulated bioreactor, allowingnutrients to flow into growing cells contained in cell pen structurewhile removing wastes and inducing recombinantly-modified organisms toproduce target molecules (e.g., proteins) at a desired stage of cellgrowth. Following induction, these cells could be shunted from the cellpen to a different region of the chip for lysis by enzymatic ormechanical means. Solubilized target molecules could then be separatedfrom cellular debris by molecular filtration units incorporated directlyon-chip.

The crude mixture of target molecules and contaminating cellularproteins and nucleic acids could then be funneled through porousmatrices of differing chemical properties (e.g., cation-exchange,anion-exchange, affinity, size-exclusion) to achieve separation. If atarget molecule were tagged with a fusion protein of a particular typeto promote solubility, it could be affinity purified, briefly treatedwith a similarly-tagged, site-specific protease to separate the fusionproduct, and then repassaged though the affinity matrix as a clean-upstep.

Once pure, the target could be mixed with different stabilizing agents,assayed for activity, and then transported to crystallization stagingareas. Localized heating (such as an electrode) and refrigeration (suchas a Peltier cooler) units stationed at various points on a chip or achip holder would allow for differential temperature regulation at allstages throughout the processing and crystallization. Thus, theproduction, purification, and crystallization of proteins may beaccomplished on an embodiment of a single microfluidics device inaccordance with the present invention.

Furthermore, since many thousands of unique solutions may be mixeddirectly on chip, the present invention may be used to do exhaustivescreening of protein crystallization conditions. This screening may bedone in a random or systematic way. Once mixed, crystallizationreactions may be routed to a locations device for storage andinspection.

III. Micro-Free Interface Diffusion

A conventional approach to crystallization has been to effect a gradualchange in target solution conditions by introducing a crystallizingagent through slow diffusion. One method that is particularly effectiveat sampling a wide range of conditions is macroscopic free-interfacediffusion. This technique requires the creation of a well-definedfluidic interface between two or more solutions, typically the proteinstock, and the precipitating agent, and the subsequent equilibration ofthe two solutions via a diffusive process. As the solutions diffuse intoone another, a gradient is established along the diffusion path, and acontinuum of conditions is simultaneously sampled. Since there is avariation in the conditions, both in space, and in time, informationregarding the location and time of crystal formation may be used infurther optimization. FIGS. 29A-29D are simplified schematic diagramsplotting concentration versus distance for a solution A and a solution Bin contact along a free interface. FIGS. 29A-D show that over time, acontinuous and broad range of concentration profiles of the twosolutions is ultimately created.

Despite the efficiency of macroscopic free-interface diffusiontechniques, technical difficulties have rendered it unsuitable for highthroughput screening applications, and it is not widely used in thecrystallographic community for several reasons. First, the fluidicinterfaces are typically established by dispensing the solutions into anarrow container; such as a capillary tube or a deep well in a cultureplate. FIGS. 30A-B show simplified cross-sectional views of theattempted formation of a macroscopic free-interface in a capillary tube9300. The act of dispensing a second solution 9302 into a first solution9304 creates convective mixing and results in a poorly defined fluidicinterface 9306.

Moreover, the solutions may not be sucked into a capillary serially toeliminate this problem. FIGS. 31A-B show the mixing, between a firstsolution 9400 and a second solution 9402 in a capillary tube 9404 thatwould result due to the parabolic velocity distribution of pressuredriven Poiseuille flow, resulting in a poorly defined fluidic interface9406. Furthermore, the container for a macro free-interfacecrystallization regime must have dimensions making them accessible to apipette tip or dispensing tool, and necessitating the use of large(10-100 μl) volumes of protein and precipitant solutions.

In order to avoid unwanted convective mixing, care must be taken bothduring dispensing and during crystal incubation. For this reasoncumbersome protocols are often used to define a macro free-interface.For example, freezing one solution prior to the addition of the second.Moreover, two solutions of differing density will mix by gravity inducedconvection if they are not stored at the proper orientation,additionally complicating the storage of reactions. This is shown inFIGS. 32A-C, wherein over time first solution 9500 having a densitygreater than the density of second solution 9502 merely sinks to form astatic bottom layer 9504 that is not conducive to formation of adiffusion gradient along the length of a capillary tube.

In accordance with embodiments of the present invention, acrystallization technique analogous to traditional macro-free interfacediffusion, called gated micro free interface diffusion (Gated μ-FID),has been developed. Gated μ-FID retains the efficient sampling of phasespace achieved by macroscopic free interface diffusion techniques,

A microfluidic free interface (μFI) in accordance with embodiments ofthe present invention is a localized interface between at least onestatic fluid and another fluid wherein mixing between them is dominatedby diffusion rather than by convective flow. For the purposes of thisapplication, the term “fluid” refers to a material having a viscositybelow a particular maximum. Examples of such maximum viscosities includebut are not limited to 1000 CPoise, 900 CPoise, 800 CPoise, 700 CPoise,600 CPoise, 500 CPoise, 400 CPoise, 300 CPoise, 250 CPoise, and 100CPoise, and therefore exclude gels or polymers containing materialstrapped therein.

In a microfluidic free interface in accordance with an embodiment of thepresent invention, at least one dimension of the interface is restrictedin magnitude such that viscous forces dominate other forces. Forexample, in a microfluidic free interface in accordance with anembodiment of the present invention, the dominant forces acting upon thefluids are viscous rather than buoyant, and hence the microfluidic freeinterface may be characterized by an extremely low Grashof number (seediscussion below). The microfluidic free interface may also becharacterized by its localized nature relative to the total volumes ofthe fluids, such that the volumes of fluid exposed to the steeptransient concentration gradients present initially after formation ofthe interface between the pure fluids is limited.

The properties of a microfluidic free interface created in accordancewith embodiments of the present invention may be contrasted with anon-free microfluidic interface, as illustrated in FIGS. 33A and 33B.Specifically, FIG. 33A shows a simplified cross-sectional view of amicrofluidic free interface in accordance with an embodiment of thepresent invention. Microfluidic free interface 7500 of FIG. 33A isformed between first fluid A and second fluid B present within channel7502. The free microfluidic interface 7500 is substantially linear, withthe result that the steep concentration gradient arising between fluidsA and B is highly localized within the channel.

As described above, the dimensions of channel 7502 are extremely small,with the result that non-slip layers immediately adjacent to the wallsof the channel in fact occupy most of the volume of the channel. As aresult, viscosity forces are much greater than buoyant forces, andmixing between fluids A and B along interface 7500 occurs almostentirely as a result of diffusion, with little or no convective mixing.

Conditions associated with the microfluidic free interface ofembodiments of the present invention can be expressed in terms of theGrashof number (Gr) per Equation (1) below, an expression of therelative magnitude of buoyant and viscous forces:

$\begin{matrix}{{{Gr} = {{B/V} = \frac{{\alpha\Delta}\; {cg}\; L^{3}}{v^{2}}}},} & (1)\end{matrix}$

where:

-   -   Gr=Grashof number;    -   B=buoyancy force;    -   V=viscous force;    -   α=solutal expansivity;    -   c=concentration;    -   g=acceleration of gravity;    -   L=chamber critical dimension; and    -   ν=kinematic viscosity.

According to Equation (1), a number of approaches may be taken to reducethe Grashof number and hence the presence of unwanted corrective flow.One such approach is to reduce g, and this is the tactic adopted bymicrogravity crystallization experiments conducted in space. Anotherapproach is to increase ν, and this is the tactic adopted byinvestigators working with gel acupuncture techniques, as describedgenerally by Garcia-Ruiz et al., “Agarose as Crystallization Media forProteins I: Transport Processes”, J. Crystal Growth 232, 165-172 (2001),hereby incorporated by reference for all purposes.

Embodiments in accordance with the present invention seek to reduce Land through the use of microfluid flow channels and vessels havingextremely small dimensions. The effect of this approach is amplified bythe cubed power of the variable (L) in Equation (1).

Microfluidic free interfaces in accordance with embodiments of thepresent invention would be expected to exhibit a Grashof number of 1 orless. The Grashof number expected with two fluids having the samedensity is zero, and thus Grashof, numbers very close to zero would beexpected to be attained.

The embodiment of a microfluidic free interface illustrated above inFIG. 33A may be contrasted with the conventional non-microfluidic freeinterface shown in FIG. 33B. Specifically, first and second fluids A andB are separated by an interface 7504 that is not uniform or limited bythe cross-sectional width of channel 7502. The steep concentrationgradient occurring at the interface is not localized, but is insteadpresent at various points along the length of the channel, exposingcorrespondingly large volumes of the fluids to the steep gradients. Inaddition, viscosity forces do not necessarily dominate over buoyancyforces, with the result that mixing of fluids A and B across interface7504 can occur both as the result of diffusion and of convective flow.The Grashof number exhibited by a conventional non-microfluidicinterface would be expected to exceed one.

1. Creation of Microfluidic Free Interface

A microfluidic free interface in accordance with embodiments of thepresent invention may be created in a variety of ways. One approach isto utilize the microfabricated elastomer structures previouslydescribed. Specifically, in certain embodiments the elastomeric materialfrom which microfluidic structures are formed is relatively permeable tocertain gases. This gas permeability property may be exploited utilizingthe technique of pressurized out-gas priming (POP) to form well-defined,reproducible fluidic interfaces.

FIG. 34A shows a plan view of a flow channel 9600 of a microfluidicdevice in accordance with an embodiment of the present invention. Flowchannel 9600 is separated into two halves by actuated valve 9602. Priorto the introduction of material, flow channel 9600 contains a gas 9604.

FIG. 34B shows the introduction of a first solution 9606 to first flowchannel portion 9600 a under pressure, and the introduction of a secondsolution 9608 to second flow channel portion 9600 b under pressure.Because of the gas permeability of the surrounding elastomer material9607, gas 9604 is displaced by the incoming solutions 9608 and 9610 andout-gasses through elastomer 9607.

As shown in FIG. 34C, the pressurized out-gas priming of flow channelportions 9600 a and 9600 b allows uniform filling of these dead-endedflow channel portions without air bubbles. Upon deactuation of valve9602 as shown in FIG. 34D, microfluidic free interface 9612 is defined,allowing for formation of a diffusion gradient between the fluids.

The formation of protein crystals utilizing gated retains the efficientsampling of phase space achieved by macroscopic free interface diffusiontechniques, with a number of added advantages, including theparsimonious use of sample solutions, ease of set-up, creation of welldefined fluidic interfaces, control over equilibration dynamics, and theability to conduct high-throughput parallel experimentation.

Another possible advantage of the formation of protein crystalsutilizing gated μ-FID is the formation of high quality crystals, asillustrated in connection with FIGS. 36A and 36B. FIG. 36A shows asimplified schematic view of a protein crystal being formed utilizing aconventional macroscopic free interface diffusion technique.Specifically, nascent protein crystal 9200 is exposed to sample fromsolution 9202 that is experiencing a net conductive flow of sample as aresult of the action of buoyant forces. As a result of thedirectionality of this conductive flow, the growth of protein crystal9200 is also directional. However, as described in Nerad et al.,“Ground-Based Experiments on the Minimization of Convection During theGrowth of Crystals from Solution”, Journal of Crystal Growth 75, 591-608(1986), assymetrical growth of a protein crystal can give rise tounwanted strain in the lattice of the growing crystal, promotingdislocations and/or the incorporation of impurities within the lattice,and otherwise adversely affecting the crystal quality.

By contrast, FIG. 36B shows a simplified schematic view of a proteincrystal being formed utilizing diffusion across a microfluidic freeinterface in accordance with an embodiment of the present invention.Nascent protein crystal 9204 is exposed to sample solution 9206 that isdiffusing within the crystallizing agent. This diffusion isnondirectional, and the growth of protein crystal 9204 is alsocorrespondingly nondirectional. Accordingly, the growing crystal avoidsstrain on the lattice and the attendant incorporation of impurities anddislocations experienced by the growing crystal shown in FIG. 36A.Accordingly, the quality of the crystal in FIG. 36B is of high quality.

While the specific embodiment just described exploits the permeabilityof the bulk material to dead end fill two or more chambers or channelsseparated by a closed valve, and creates a microfluidic free interfacebetween the static fluids by the subsequent opening of this valve, othermechanisms for realizing a microfluidic free interface are possible.

For example, FIG. 46 shows one potential alternative method forestablishing a microfluidic free interface diffusion in accordance withthe present invention. Microfluidic channel 8100 carries fluid Aexperiencing a convective flow in the direction indicated by the arrow,such that static non-slip layers 8102 are created along the walls offlow channel 8100. Branch channel 8104 and dead-ended chamber 8106contain static fluid B. Because material surrounding the dead-endedchannel and chamber provide a back pressure, fluid B remains static andmicrofluidic free interface 8110 is created at mouth 8112 of branchchannel 8108 between flowing fluid A and static fluid B. As describedbelow, diffusion of fluid A or components thereof across themicrofluidic free interface can be exploited to obtain useful results.While the embodiment shown in FIG. 46 includes a dead-ended branchchannel and chamber, this is not required by the present invention, andthe branch channel could connect with another portion of the device, aslong as a sufficient counter pressure was maintained to prevent any netflow of fluid through the channel.

Another potential alternative method for establishing a microfluidicfree interface diffusion assay is the use of break-through valves andchambers. A break-through valve is not a true closing valve, but rathera structure that uses the surface tension of the working fluid to stopthe advance of the fluid. Since these valves depend on the surfacetension of the fluid they can only work while a free surface exists atthe valve; not when the fluid continuously fills both sides and theinterior of the valve structure.

A non-exclusive list of ways to achieve such a valve include but are notlimited to patches of hydrophobic material, hydrophobic treatment ofcertain areas, geometric constrictions (both in height and width) of achannel, geometric expansions (both in height and in width of achannel), changes in surface roughness on walls of a channel, andapplied electric potentials on the walls.

These “break-through” valves may be designed to withstand a fixed andwell defined pressure before they “break through” and allow fluid topass nearly unimpeded. The pressure in the channel can be controlled andhence the fluid can be caused to advance when desired. Different methodsof controlling this pressure include but are not limited to externallyapplied pressure at an input or output port, pressure derived fromcentrifugal force (i.e. by spinning the device), pressure derived fromlinear acceleration (i.e. applying an acceleration to the device with acomponent parallel to the channel), elecrokinetic pressure, internallygenerated pressure from bubble formation (by chemical reaction or byhydrolysis), pressure derived from mechanical pumping, or osmoticpressure.

“Break-through” valves may be used to create a microfluidic freeinterface as shown and described in connection with FIGS. 35A-E. FIG.35A shows a simplified plan view of a device for creating a microfluidicfree interface utilizing break through valves. First chamber 9100 is influid communication with second chamber 9102 through branches 9104 a and9104 b respectively, of T-shaped channel 9104.

First break through valve 9106 is located at outlet 9108 of firstchamber 9100. Second break through valve 9110 is located in branch 9104b upstream of inlet 9105 of second chamber 9102. Third break throughvalve 9112 is located at outlet 9114 of second chamber 9102.Breakthrough valves 9106, 9110, and 9112 may be formed from hydrophobicpatches, a constriction in the width of the flow channel, or some otherway as described generally above. In FIGS. 35A-E, an open break throughvalve is depicted as an unshaded circle, and a closed break throughvalve is depicted as a shaded circle.

In the initial stage shown in FIG. 35B, first chamber 9100 is chargedwith first fluid 9116 introduced through stem 9104 c and branch 9104 aof T-shaped channel 9104 and chamber inlet 9107 at a pressure below thebreak through pressure of any of the valves 9106, 9110, and 9112. In thesecond stage shown in FIG. 35C, second chamber 9102 is charged with abuffer or other intermediate fluid 9118 introduced through stem 9104 cof T-shaped channel 9104 and inlet 9105 at a pressure below the breakthrough pressure of valve 9106 but greater than the break throughpressures of valves 9110 and 9112.

In the third stage shown in FIG. 35D, intermediate fluid 9118 isreplaced in second chamber 9102 with second fluid 9120 introducedthrough stem 9104 c of T-shaped channel 9104 and inlet 9105 at apressure below the break through pressure of valve 9106 but greater thanthe break through pressures of valves 9110 and 9112. In the final stagedepicted in FIG. 35E, second fluid 9120 has replaced the intermediatefluid, leaving the first and second fluids 9116 and 9120 in separatechambers but fluidically connected through T-junction 9104, creating amicrofluidic free interface 9122.

The use of break through valves to create a microfluidic free interfacein accordance with embodiments of the present invention is not limitedto the specific example given above. For example, in alternativeembodiments the step of flushing with a buffer or intermediate solutionis not required, and the first solution could be removed by flushingdirectly with the second solution, with potential unwanted by-productsof mixing removed by the initial flow of the second solution through thechannels and chambers.

While the embodiments just described create the microfluidic freeinterface in a closed microfluidic device, this is not required byembodiments in accordance with the present invention. For example, analternative embodiment in accordance with the present invention mayutilize capillary forces to connect two reservoirs of fluid. In oneapproach, the open wells of a micro-titer plate could be connected by asegment of a glass capillary. The first solution would be dispensed intoone well such that it fills the well and is in contact with the glasscapillary. Capillary forces cause the first solution to enter and flowto the end of the capillary. Once at the end, the fluid motion ceases.Next, the second solution is added to the second well. This solution isin contact with the first solution at the capillary inlet and creates amicrofluidic interface between the two wells at the end of thecapillary.

The connecting path between the two wells need not be a glass capillary,and in alternative embodiments could instead comprise a strip ofhydrophilic material, for example a strip of glass or a line of silicadeposited by conventional CVD or PVD techniques. Alternatively, theconnecting paths could be established by paths of less hydrophobicmaterial between patterned regions of highly hydrophobic material.Moreover, there could be a plurality of such connections between thewells, or a plurality of interconnected chambers in variousconfigurations. Such interconnections could be established by the userprior to use of the device, allowing for rapid and efficient variationin fluidic conditions.

Where as in the previous example the two reservoirs are not enclosed bya microfluidic device but are connected instead through a microfluidicpath, an alternative embodiment could have reservoirs both enclosed andnot enclosed. For example, sample could be loaded into a microfluidicdevice and pushed to the end of an exit capillary or orifice (by any ofthe pressure methods described above). Once at the end of the exitcapillary, the capillary could be immersed in a reservoir of reagent. Inthis way, the microfluidic free interface is created between theexternal reservoir and the reservoir of reagent in the chip. This methodcould be used in parallel with many different output capillaries ororifices to screen a single sample against a plurality of differentreagents using microfluidic free interface diffusion.

In the example just described, the reagent is delivered from one or manyinlets to one or many different outlets “through” a microfluidic device.Alternatively, this reagent can be introduced through the same orificethat is to be used to create the microfluidic interface. Thesample-containing solution could be aspirated into a capillary (eitherby applying suction, or by capillary forces, or by applying pressure tothe solution) and then the capillary may be immersed in a reservoir ofcounter-reagent, creating a microfluidic interface between the end ofthe capillary and the reservoir. This could be done in a large array ofcapillaries for the parallel screening of many different reagents. Verysmall volumes of sample could be used since the capillaries can have afixed length beyond which the sample will not advance. Forcrystallization applications (see below), the capillaries could beremoved and mounted in an x-ray beam for diffraction studies, withoutrequiring handling of the crystals.

2. Reproducible Control Over Equilibration Parameters

One advantage of the use of microfluidic free interface diffusion inaccordance with embodiments of the present invention is the ability tocreate uniform and continuous concentration gradients that reproduciblysample a wide range of conditions. As the fluids on either side of theinterface diffuse into one another, a gradient is established along thediffusion path, and a continuum of conditions is simultaneously sampled.Since there is a variation in the conditions, both in space and time,information regarding the location and time of positive results (i.e.crystal formation) may be used in further optimization.

In many applications it is desirable to create a gradient of a conditionsuch as pH, concentration, or temperature. Such gradients may be usedfor screening applications, optimization of reaction conditions,kinetics studies, determination of binding affinities, dissociationconstants, enzyme-rate profiling, separation of macromolecules, and manyother applications. Due principally to the suppression of convectiveflow, diffusion across a microfluidic free interface in accordance withan embodiment of the present invention may be used to establish reliableand well-defined gradient.

The dimensional Einstein equation (2) may be employed to obtain a roughestimate of diffusion times across a microfluidic free interface.

$\begin{matrix}{{t = \frac{x^{2}}{4D}};} & (2)\end{matrix}$

where:

-   -   t=diffusion time;    -   x=longest diffusion length; and    -   D=diffusion coefficient        Generally, as shown in Equation (3) below, the diffusion        coefficient varies inversely with the radius of gyration, and        therefore as one over the cube root of molecular weight.

$\begin{matrix}{{D \propto \frac{1}{r} \propto \frac{1}{m^{1/3}}};} & (3)\end{matrix}$

where:

-   -   D=diffusion coefficient;    -   r=radius of gyration; and    -   m=molecular weight

In reviewing equation (3), it is important to recognize that correlationbetween the radius of gyration (r) and the molecular weight (m) is onlyan approximation. Because of the dominance of viscous forces overinertial forces, the diffusion coefficient is in fact independent ofmolecular weight and is instead dependent upon the size and hence dragexperienced by the diffusing particle.

As compared with the rough 1.5 hr equilibration time for a dye, anapproximate equilibration time for a protein of 20 KDa over the samedistance is estimated to be approximately 45 hours. The equilibrationtime for a small salt of a molecular weight of 100 Da over the samedistance is about 45 minutes.

The relative concentrations resulting from diffusion across a fluidicinterface is determined not only by thermodynamic conditions exploredduring the equilibration, but also by the rate at which equilibrationtakes place. It is therefore potentially valuable to control thedynamics of equilibration.

In conventional macroscopic diffusion methods, only coarse control overthe dynamics of equilibration may be available through manipulation ofinitial conditions. For macroscopic free interface diffusion, oncediffusion begins, the experimenter has no control over the subsequentequilibration rate. For hanging drop experiments, the equilibration ratemay be changed by modifying the size of the initial drop, the total sizeof the reservoir, or the temperature of incubation. In microbatchexperiments, the rate at which the sample is concentrated may be variedby manipulating the size of the drop, and the identity and amount of thesurrounding oil. Since the equilibration rates depend in a complicatedmanner on these parameters, the dynamics of equilibration may only bechanged in a coarse manner. Moreover, once the experiment has begun, nofurther control over the equilibration dynamics is available.

By contrast, in a fluidic free interface experiment in accordance withan embodiment of the present invention, the parameters of diffusiveequilibration rate may also be controlled by manipulating dimensions ofchambers and connecting channels of a microfluidic structure. Forexample, in a microfluidic structure comprising reservoirs in fluidcommunication through a constricted channel, where no appreciablegradient exists in the reservoirs due to high concentrations orreplenishment of material, to good approximation the time required forequilibration varies linearly with the required diffusion length. Theequilibration rate also depends on the cross-sectional area of theconnecting channels. The required time for equilibration may thereforebe controlled by changing both the length, and the cross-sectional areaof the connecting channels.

For example, FIG. 40A shows a plan view of a simple embodiment of amicrofluidic structure in accordance with the present invention.Microfluidic structure 9701 comprises reservoirs 9700 and 9702containing first fluid A and second fluid B, respectively. Reservoirs9700 and 9702 are connected by channel 9704. Valve 9706 is positioned onthe connecting channel between reservoirs 9700 and 9702.

Connecting channel 9704 has a much smaller cross-sectional area thaneither of the reservoirs. For example, in particular embodiments ofmicrofluidic structures in accordance with the present invention, theratio of reservoir/channel cross-sectional area and thus the ratio ofmaximum ratio of cross-sectional area separating the two fluids, mayfall between 500 and 25,000. The minimum of this range describes a50×50×50 μm chamber connected to a 50×10 μm channel, and the maximum ofthis range describes a 500×500×500 μm chamber connected to a 10×1 μmchannel.

Initially, reservoirs 9700 and 9702 are filled with respective fluids,and valve 9706 is closed. Upon opening valve 9706, a microfluidic freeinterface in accordance with an embodiment of the present invention iscreated, and fluids A and B diffuse across this interface through thechannel into the respective reservoirs. Moreover, where the amount ofdiffusing material present in one reservoir is large and the capacity ofthe other reservoir to receive material without undergoing a significantconcentration change is also large, the concentrations of material inthe reservoirs will not change appreciably over time, and a steady stateof diffusion will be established.

Diffusion of fluids in the simple microfluidic structure shown in FIG.40 may be described by relatively simple equations. For example, the netflux of a chemical species from one chamber to the other may be simplydescribed by equation (4):

$\begin{matrix}{{J = {D*A*\frac{\Delta \; C}{L}}};} & (4)\end{matrix}$

where:

-   -   J=net flux of chemical species    -   D=diffusion constant of the chemical species;    -   A=cross-sectional area of the connecting channel;    -   ΔC=concentration difference between the two channels; and    -   L=length of the connection channel.

Following integration and extensive manipulation of the terms ofequation (4), the characteristic time τ for the equilibration of the twochambers, where one volume V₁ is originally at concentration C and theother volume V₂ is originally at concentration 0, can therefore be takento be as shown in Equation (5) below:

$\begin{matrix}{{\tau = {\frac{1}{{V_{1}/V_{2}} + 1}*\frac{1}{D}*\frac{L}{A/V_{1}}}};} & (5)\end{matrix}$

where:

-   -   τ=equilibration time;    -   V₁=volume of chamber initially containing the chemical species;    -   V₂=volume of chamber into which the chemical species is        diffusing;    -   D=diffusion constant of the chemical species;    -   A=cross-sectional area of the connecting channel; and    -   L=length of the connection channel.

Therefore, for a given initial concentration of a chemical species in achamber of a defined volume, the characteristic equilibration timedepends in a linear manner from the diffusive length L and the ratio ofthe cross-sectional area to the volume (hereafter referred to simply asthe “area”), with the understanding that the term “area” refers to thearea normalized by the volume of the relevant chamber. Where twochambers are connected by a constricted channel, as in the structure ofFIG. 40A, the concentration drop from one channel to the other occursprimarily along the connecting channel and there is no appreciablegradient present in the chamber. This is shown in FIG. 40B, which is asimplified plot of concentration versus distance for the structure ofFIG. 40A.

The behavior of diffusion between the chambers of the microfluidicstructure of FIG. 40A can be modeled, for example, utilizing the PDEtoolbox of the MATLAB® software program sold by The MathWorks Inc. ofNatick, Mass. FIGS. 41 and 42 accordingly show the results of simulatingdiffusion of sodium chloride from a 300 um×300 um×100 um chamber toanother chamber of equal dimensions, through a 300 um long channel witha cross-sectional area of 1000 um. The initial concentrations of thechambers are 1 M and 0 M, respectively.

FIG. 41 plots the time required for the concentration in one of thereservoirs to reach 0.6 of the final equilibration concentration, versuschannel length. FIG. 41 shows the linear relationship between diffusiontime and channel length for this simple microfluidic system.

FIG. 42 plots the inverse of the time required for the concentration inone of the reservoirs to reach 0.6 of the final equilibrationconcentration (T_(0.6)), versus the area of the fluidic interfacecreated upon opening of the valve. FIG. 42 shows the linear relationshipbetween these parameters. The simple relationship between theequilibration time constant and the parameters of channel length and1/channel area allows for a reliable and intuitive method forcontrolling the rate of diffusive mixing across a microfluidic freeinterface in accordance with an embodiment of the present invention.

This relationship further allows for one reagent to be diffusively mixedwith a plurality of others at different rates that may be controlled bythe connecting channel geometry. For example, FIG. 38A shows three setsof pairs of compound chambers 9800, 9802, and 9804, each pair connectedby microchannels 9806 of a different length Δx. FIG. 38B plotsequilibration time versus equilibration distance. FIG. 38B shows thatthe required time for equilibration of the chambers of FIG. 38A variesas the length of the connecting channels.

FIG. 39 shows four compound chambers 9900, 9902, 9904, and 9906, eachhaving different arrangements of connecting microchannel(s) 9908.Microchannels 9908 have the same length, but differ in cross-sectionalarea and/or number of connecting channels. The rate of equilibration maythus be increased/decreased by decreasing/increasing the cross-sectionalarea, for example by decreasing/increasing the number of connectingchannels or the dimensions of those channels.

Another desirable aspect of microfluidic free interface diffusionstudies in accordance with embodiments of the present invention is theability to reproducibly explore a wide range of phase space. Forexample, it may be difficult to determine, a priori, which thermodynamicconditions will be favorable for a particular application (i.e.nucleation/growth of protein crystals), and therefore it is desirablethat a screening method sample as much of phase-space (as manyconditions) as possible. This can be accomplished by conducting aplurality of assays, and also through the phase space sampled during theevolution of each assay in time.

FIG. 43 shows the results of simulating the counter-diffusion oflysozyme and sodium chloride utilizing the microfluidic structure shownin FIG. 40, with different relative volumes of the two reservoirs, andwith initial concentrations normalized to 1. FIG. 43 presents a phasediagram depicting the phase space between fluids A and B, and the pathin phase space traversed in the reservoirs as the fluids diffuse acrossthe microfluidic free interface created by the opening of the valve inFIG. 40. FIG. 43 shows that the phase space sampled depends upon theinitial relative volumes of the fluids contained in the two reservoirs.By utilizing arrays of chamber with different sample volumes, and thenidentifying instances where diffusion across the channel yieldeddesirable results (i.e. crystal formation), promising starting pointsfor additional experimentation can be determined.

As described above, varying the length or cross-sectional area of achannel connecting two reservoirs changes the rate at which the speciesare mixed. However, so long as the channel volume remains small comparedas compared with the total reaction volume, there is little or no effecton the evolution of concentration in the chambers through phase space.The kinetics of the mixing are therefore decoupled from the phase-spaceevolution of the reaction, allowing the exercise independent controlover the kinetic and thermodynamic behavior of the diffusion.

For example, it is often desirable in crystallography to slow down theequilibration so as to allow for the growth of fewer and higher qualitycrystals. In conventional techniques this is often attempted by addingnew chemical constituents such as glycerol, or by using microbatchmethods. However, this addition of constituents is not wellcharacterized, is not always effective, and may inhibit the formation ofcrystals. Microbatch methods also may pose the disadvantage of lacking adriving force to promote continued crystal growth as protein in thesolution surrounding the crystal is depleted. Through the use ofdiffusion across a microfluidic free interface in accordance with anembodiment of the present invention, crystal formation may be slowed bya well-defined amount without altering the phase-space evolution, simplyby varying the width or cross-sectional area of the connecting channel.

The ability to control the rate at which equilibration proceeds hasfurther consequences in cases were one wishes to increase the totalvolume of a reaction while conserving both the thermodynamics and themicrofluidic free interface diffusion mixing. One such case arises againin the context of protein crystallography, in which an initial, smallvolume crystallization assay results in crystals of insufficient sizefor diffraction studies. In such a case, it is desirable to increase thereaction volume and thereby provide more protein available for crystalgrowth, while at the same time maintaining the same diffusive mixing andpath through phase space. By increasing the chamber volumesproportionally and decreasing the area of the channel, the area of theinterface relative to the total assay volume is reduced, and a largervolume would pass through the same phase space as in the original smallvolume conditions.

While the above description has focused upon diffusion of a singlespecies, gradients of two or more of species which do not interact witheach other may be created simultaneously and superimposed to create anarray of concentration conditions. FIG. 47 shows a plan view of oneexample of a microfluidic structure for creating such superimposedgradients. Flat, shallow chamber 8600 constricted in the verticaldirection is connected at its periphery to reservoirs 8602 and 8604having fixed concentrations of chemical species A and B, respectively.Sink 8606 in the form of a reservoir is maintained at a substantiallylower concentration of species A and B. After the initial transientequilibration, stable and well-defined gradients 8608 and 8610 ofspecies A and B respectively, are established in two dimensions.

As evident from inspection of FIG. 47, the precise shape and profile ofthe concentration gradient will vary according to a host of factors,including but not limited to the relative location and number of inletsto the chamber, which can also act as concentration sinks for thechemical species not contained therein (i.e. reservoir 8604 may act as asink for chemical species A). However, the spatial concentrationprofiles of each chemical species within the chamber may readily bemodeled using the MATLAB program previously described to describe atwo-dimensional, well-defined, and continuous spatial gradient.

The specific embodiment illustrated in FIG. 47 offers the disadvantageof continuous diffusion of materials. Hence, where diffusion of productsof reaction between the diffusing species is sought to be discerned,these products will themselves diffuse in the continuous gradient,thereby complicating analysis.

Accordingly, FIG. 48 shows a simplified plan view of an alternativeembodiment of a microfluidic structure for accomplishing diffusion intwo dimensions. Grid 8700 of intersecting orthogonal channels 8702establishes a spatial concentration gradient. Reservoirs 8704 and 8708of fixed concentrations of chemical species A and B are positioned onadjacent edges of grid 8700. Opposite to these reservoirs on the gridare two sinks 8703 of lower concentration of the chemical present in theopposing reservoirs.

Surrounding each channel junction 8710 are two pairs of valves 8712 and8714 which control diffusion through the grid in the vertical andhorizontal directions, respectively. Initially, only valve pairs 8714are opened to create a well-defined diffusion gradient of the firstchemical in the horizontal direction. Next, valve pairs 8714 are closedand valve pairs 8712 opened to create a well-defined diffusion gradientof the second chemical in the vertical direction. Isolated by adjacenthorizontal valves, the gradient of the first chemical species remainspresent in regions between the junctions.

Once the second (vertical) gradient is established, the two gradientscan be combined and by opening all the valve pairs for a short time toallow partial diffusive equilibration. After the period of diffusion haspassed, all the valve pairs are closed to contain the superimposedgradient. Alternatively, valve pairs 8712 and 8714 can be closed to haltdiffusion in the vertical direction, with every second horizontal valveopened to create separate isolated chambers.

To summarize, conventional macro free-interface techniques employcapillary tubes or other containers having dimensions on the order ofmm. By contrast, the fluidic interface in accordance with embodiments ofthe present invention is created in a microchannel having dimensions onthe order of μm. At such small dimensions, unwanted convection issuppressed due to viscosity effects, and mixing is dominated bydiffusion. A well-defined fluidic interface may thus be establishedwithout significant undesirable convective mixing.

IV. Crystal Storage and Analysis

Combining the basic metering and mixing functionality with a fluidicstorage structure, allows for a complete protein crystallizationworkstation to be implemented on chip. In this way a researcher mayexplore the solubility of a protein in various chemistries, decide whichare the most promising crystallization conditions, and then set andincubate reactions for crystal growth. In this way, screening, phasespace exploration, optimization, and incubation may be achieved on asingle microfluidic workstation. A non-exclusive list of possiblemethods of storage is provided below.

In accordance with one embodiment of the present invention, reactionsmay be stored by pumping pre-mixed reagent (crystallizing agents,additives, cryo-protectants . . . , sample) into a storage channel andseparating the experiments by an immiscible fluid (eg. Paraffin oil).FIGS. 58A-C are schematic views of one implementation of this storagetechnique.

In FIG. 58A, a sample is flowed into circular mixing device 5800 throughthe peristaltic pumping action of serial valves 5802 a-c. Mixture 5803is then created and flowed into cross junction 5804. In FIG. 58B, thesample within cross-junction 5804 is routed to serpentine, storagechannel 5806 having end 5806 a controlled by valve 5810. In FIG. 58C,the valve configuration is changed, and an inert separating fluid 5808such as oil is flowed into cross junction 5804. In FIG. 58D, theseparating fluid 5808 is flowed into storage channel 5806. The cycleillustrated in FIGS. 58A-D may then be repeated to provide a new samplevolume within storage line 5806. Samples stored within channel 5806 maybe recovered through end 5806 a through gate valve 5810.

Assuming that the storage channel has dimensions 100 μm wide * 100 μmtall, a 1 nL sample would fill a length of channel equal to 100 μm.Assuming that the channel is serpentine and that adjacent legs areseparated by 100 μm, the total length of channel that would fit on a lcmsquare storage area is approximately 1 cm * 100/2=0.5 m. This wouldallow the storage of 0.5 m/100 μm=5000 reactions.

Since the entire length of fluid must be advanced for every addition, itmay prove difficult to pump this long length of fluid. To avoid thisproblem, FIG. 59 shows a schematic view of embodiment 5900 of areaction/storage scheme wherein multiplexer 5902 could be used to directan experimental sample and inert separating fluid into one of aplurality of parallel storage channels 5904.

In accordance with still another alternative embodiment, each reactioncould be dead-end filled to the end of the storage line so that theentire column of fluid need never be moved together. FIG. 60 shows asimplified schematic view of such an approach, wherein storage channel6000 is dead-ended.

A flow of air could be utilized to bias the samples and inert separatingliquid into the storage channel, with the air ultimately diffusing outof the channel through the elastomer material. In such an embodiment,the relatively high pressures required to accomplish dead-ended fillingcould be achieved using an external pressure source, thereby eliminatingthe need for a separate pump on the oil line. This dead-end fillingtechnique could be used to fill a single storage line as in theembodiment shown in FIGS. 58A-D, or many parallel storage lines througha multiplexer as in the embodiment shown in FIG. 59.

While FIGS. 58A-60 show the storage line as being integrated in a planarfashion as a channel on the chip, this is not required by the presentinvention. In accordance with alternative embodiments of the presentinvention the reagents may be off-loaded from the chip in the verticaldirection, for example into a glass capillary.

Still another approach for storing chemicals is to utilize diffusionassays. FIGS. 54A-D show a layout of a device that combines combinatoricmixing structure 5400 with an addressable storage array 5402. Array 5402allows for incubation on chip of 256 individual batch experiments or 128free interface diffusion experiments.

FIG. 54B shows a blow-up of the entrance to storage array 5402. Array5402 works on the dead-end loading principal discussed above. Once thereagents are mixed in ring 5406, they are pumped into serpentine channel5408 for temporary storage. Multiplexer 5410 at the storage array inletis then actuated to open one of the sixteen possible inlets, therebyselecting the array row to be addressed.

FIG. 54C shows an enlarged view of the storage array of FIG. 54A. FIG.54D shows an enlarged view of a single cell in the array of FIG. 54D.

Each row of the storage array has a control line 5452 that actuatesvalves 5454 separating storage chambers 5456 from the channel inlets,and a control line 5458 that separates the columns of the storage array.A single control line 5460 is further routed to every pair offluidically coupled chambers 5462 a-b to separate them until it isdesired to create a fluidic interface.

Once the row is selected by the multiplexer, the array column 5470 isselected by actuating a corresponding column valve 5472. In this way asingle chamber of the array is selected for filling.

Valves near the outlet of the ring are actuated to connect theserpentine storage line to the multiplexer inlet, and the storedsolution is pushed back out of the serpentine storage line and into themultiplexer area by pressurized air. This pressurization drives thefluid into the appropriate row of the storage array, pressurizing theair ahead of it and causing it to diffuse into the polymer.

While the chamber inlet valves remain closed, the fluid does not enterthe chamber, but rather remains in the dead volume between themultiplexer and the storage array (or partly in this volume and partlyin the storage array channels). A new line of the multiplexer is thenselected and the column valve is temporarily opened to allow the new rowto be flushed with buffer as a precaution to avoid cross contamination.Since only one line of the multiplexer is open the other rows of thestorage array are held fixed.

This new row is then emptied by blowing air through it, preparing it forthe next solution. These steps are repeated until all rows are filledwith a unique solution.

The column valve is then actuated, and the inlet valves opened, and allrows are simultaneously pressurized. This drives the solutions intotheir respective chambers. This entire process can be repeated for everycolumn until the array is filled with solutions (potentially a differentsolution in every chamber).

If the interface valves are held closed, the array of FIGS. 54A-D willaccommodate 256 (8 columns×16 rows) batch reactions. In applicationssuch as protein crystallography where diffusion across a microfluidicfree interface is desired, the interface valves may be opened tocommence the reactions. Since all solutions have previously beenseparately mixed in the ring, the experimenter has control over thesolutions.

For example, free-interface diffusion experiments for crystallizationmay be conducted in which one or more of the following is varied:identity and/or initial concentration of the precipitating agent;identity and/or initial concentration of the crystallized species;identity and/or initial concentration of additives; and identity and/orinitial concentration of cryo-protectants. The ability to mix a host ofdifferent agents into small volumes of protein solution prior to freeinterface diffusion experiments offers an important advantage overconventional crystallization approaches, where typically a standardprotein stock is used against different crystallization agents. Themicrofluidic network described above thus offers a flexible platform forcrystallization.

The array of FIGS. 54A-D also allows for sample recovery, addressablewell flushing, and the reusing of reaction chambers. Specifically, torecover sample or flush a well the appropriate inlet valves are openedon the column that has the well to be emptied/flushed. One of the rowsof the multiplexer that connects one of the chambers of the pair to beflushed is selected.

The array row that connects the pair that was not opened at themultiplexer is then opened at the end of the array, and the row that wasopened at the multiplexer is closed at the end of the array.

This manipulation causes and open fluidic path through the selected rowof the multiplexer, through the chamber pair to be emptied/flushed, andout the row selected at the outlet. In this way a single chamber paircan be addressed and flushed.

As previously described, once a protein crystal has been formed,information about its three dimensional structure can be obtained fromdiffraction of x-rays by the crystal. However, application of highlyenergetic radiation to the protein tends to generate creates heat.X-rays are also ionizing, and can result in the production of freeradicals and broken covalent bonds. Either heat or ionization maydestroy or degrade the ability of a crystal to diffract incident x-rays.

Accordingly, upon formation of a crystal a cryogenic material istypically added to preserve the crystalline material in its alteredstate. However, the sudden addition of cryogen can also damage acrystal. Therefore, it would be advantageous for an embodiment of acrystallization chip in accordance with the present invention to enablethe direct addition of cryogen to the crystallization chamber once acrystalline material is formed therein.

In addition, protein crystals are extremely delicate, and can quicklycrumble or collapse in response to physical trauma. Accordingly,harvesting a crystal unharmed from the small chambers of a chip poses apotential obstacle to obtaining valuable information about thecrystalline material.

Therefore, it would also be advantageous for an alternative embodimentof a crystallization chip in accordance with the present invention toallow direct interrogation by x-ray radiation of crystalline materialsformed in a chip, thereby obviating entirely the need for separatecrystal harvesting procedures.

Accordingly, FIG. 53A shows a plan view of a simplified embodiment of acrystal growing chip in accordance with the present invention. FIG. 53Bshows a simplified cross-sectional view of the embodiment of the crystalgrowing chip shown in FIG. 53A along line B-B′.

Harvesting/growing chip 9200 comprises elastomer portion 9202 overlyingglass substrate 9204. Glass substrate 9204 computes three etched wells9206 a, 9206 b and 9206 c. Placement of elastomer portion 9202 overglass substrate 9204 thus defines three corresponding chambers in fluidcommunication with each other through flow channels 9208. The flow ofmaterials through flow channels 9208 is controlled by valves 9210defined by the overlap of control lines 9212 over control channels 9208.

During operation of growth/harvesting chip 9200, valves 9210 areinitially activated to prevent contact between the contents of chambers9206 a, 9206 b and 9206 c. Chambers 9206 a, 9206 b and 9206 c are thenseparately charged through wells 9214 with different materials foreffecting crystallization. For example, chamber 9206 a may be chargedwith a protein solution, chamber 9206 b may be charged with acrystallizing agent, and chamber 9206 c may be charged with a cryogen.

The first control line 9212 may then be deactivated to open valve 9210a, and thereby allowing diffusion of protein solution and crystallizingagent. Upon formation of a crystal 9216, the remaining control lines9212 may be deactivated to allow the diffusion of cryogen from chamber9206 c to preserve the crystal 9216.

Next, the entire chip 9200 may be mounted in an x-ray diffractionapparatus, with x-ray beam 9218 applied from source 9220 against crystal9216 with diffraction sensed by detector 9222. As shown in FIG. 53B, thegeneral location of wells 9206 corresponds to regions of reducedthickness of both elastomer portion 9202 and underlying glass portion9204. In this manner, radiation beam 9218 is required to traverse aminimum amount of elastomer and glass material prior to and subsequentto encountering crystal 9216, thereby reducing the deleterious effect ofnoise on the diffracted signal received.

While one example of a protein growth/harvesting chip has been describedabove in connection with FIGS. 53A-B, embodiments in accordance with thepresent invention are not limited to this particular structure. Forexample, while the embodiment of the current embodiment that isdescribed utilizes a glass substrate in which microchambers have beenetched, fabrication of microfluidic structures in accordance with thepresent invention is not limited to the use of glass substrates.Possible alternatives for fabricating features in a substrate includeinjection molding of plastics, hot embossing of plastics such as PMMA,or fabricating wells utilizing a photocurable polymer such as SU8photoresist. In addition, features could be formed on a substrate suchas glass utilizing laser ablation, or features could be formed byisotropic or aniosotropic etching of a substrate other than glass, suchas silicon.

Potential advantages conferred by alternative fabrication methodsinclude but are not limited to, more accurate definition of featuresallowing for more dense integration, and ease of production (e.g. hotembossing). Moreover, certain materials such as carbon based plasticsimpose less scattering of X-rays, thereby facilitating collection ofdiffraction data directly from a chip.

An additional possibility for the harvesting of crystals is to have amethod of off-loading from chip. Off-loading could be performed oncecrystals have formed, or alternatively, prior to incubation. Theseoff-loaded crystals could then be used to seed macroscopic reactions, orbe extracted and mounted in a cryo-loop. If a method for the addition ofcryogen was also developed, the crystals could be flash frozen andmounted directly into the x-ray beam.

Protein crystals grown in relatively small volumes (approximately 10 nL)may be extracted from chip and used in diffraction studies. The smallvolumes of protein sample used in each reaction on these devices,however, may limits the size of the protein crystals grown, making themunsuitable for high resolution diffraction studies. Furthermore, sinceprotein crystals are often very fragile, it is technically difficult toharvest crystals without damaging them. Finally, since the unique mixingobtained in the microfluidic environment enables the higher success rateof crystallization, it is not necessarily a straight forward process toreproduce crystallization conditions in a traditional large (microliter)volume format.

Accordingly, an alternative embodiment of the present invention providesa large volume crystal growth format that has a high correspondence withthe previously reported microfluidic screening chip. This deviceaddresses the need to grow larger crystals and facilitates thatharvesting of crystals from the chip.

The device of this embodiment is a made from an elastomer material andconsists of a series of microfluidic chambers connected in pairs viamicrofluidic channels. Active valves are used to separated the wells ofeach pair during loading and to isolate the pairs from the rest of thechip during incubation and crystal growth. An autoCAD drawing of a groupof five chamber pairs is shown in FIG. 17.

Operation of the device of FIG. 17 is identical to that of screeningchip described previously. This embodiment is distinct in that thevolume of the chambers has been greatly increased, and in that the wellsmay be easily made accessible by cutting and removing a thin piece ofelastomer on the bottom of the device.

The dimensions of the wells (in microns) for this embodiment are shownin FIG. 17. The depth of the wells is approximately 100 microns. Thetotal reaction volume for this design is approximately 500 nL, allowingfor the growth of protein crystals suitable for diffraction studies.Since the mixing of the reagents is still accomplished via diffusionacross a constricted channel, the kinetics that gave crystallization inthe original device are conserved, and a high degree of correspondencewith the screening chip is achieved.

FIG. 18 shows a micrograph of a crystal grown and harvested utilizingthe device of FIG. 17. The crystal shown in FIG. 18 is a large (450 kDa)complex comprising the DnaB replicative helicase protein and the DnaChelicase loader protein.

Protein crystal growth devices in accordance with embodiments of thepresent invention can be fabricated using at least two methods. In thefirst method, a multilayer elastomer chip having buried control channelsand open flow channels (approximately 20 microns high) is sealed to athin (approx 1 mm) layer of elastomer with molded cavities(approximately 200 microns high). The control channels may be actuatedto pinch off the underlying flow layer. The seal between the flow andwell layer is not permanent, so that when crystals are identified, thethin well layer may be cut with a blade and a section pealed away fromthe device to expose the crystals. Once the crystals are exposed, asolution containing a cryo-protectant may be added. The crystals arethen looped out and frozen using conventional techniques.

According to a second fabrication method, the wells are molded on thesame layer as the flow channels from a negative master having both flowfeatures (approximately 20 microns high) and well features(approximately 200 microns high). This layer is bonded on top of acontrol structure, so that the pressurization of the control channelscauses a membrane to deflect up into the flow channel, closing it. Thecontrol layer is sealed to a blank membrane that can be cut to exposethe crystals for harvesting.

Embodiments in accordance with the present invention can be made with avariety of chamber sizes, aspect ratios, channel geometries, mixingratios, number of inlets, and thicknesses. By making the devicesufficiently thin, it is possible to collect diffraction data throughthe chip without significant x-ray scattering or attenuation. By addingmore fluidics it is possible to do more advanced functions, includingbut not limited to control over the addition of cryo-protectant orligands, seeding experiments, or protein replenishment.

Once a crystallization condition is identified in the screening chip, amethod must be found to determine if the crystals diffract, optimize thegrowth conditions, grow crystals of sufficient size, addcryo-protectants or heavy metals, harvest them, get them into an x-raybeam, and collect data over a significant angle of rotation. Variousproblems and requirements which may be associated with this process aredescribed below.

First, crystallization conditions discovered in the microfluidic deviceare not necessarily transportable to conventional large-scale formats.This is because the unique mixing kinetics achieved on chip can not bereproduced in conventional formats. Therefore a technique must preservethe kinetics of microfluidic mixing while providing larger reactionvolumes. Moreover, the chip typically undergoes dehydration during thecourse of incubation that is not well controlled and thus difficult toreproduce in larger formats. Therefore there must be a technique toscreen for various levels of dehydration in the growth device.

Second, protein crystals are often very fragile and the manipulationsrequired during conventional harvesting, or harvesting from the“Microfluidic Protein Crystal Growth Chip” can damage them.

Third, although it is advantageous to freeze a crystal during datacollection, the freezing process may damage the crystals. Therefore ageneral technique is required to facilitate mounting of crystals at roomtemperature and at cryo temperatures.

Fourth, the freezing of a protein crystal requires the presence of acryo-protectant so that the solvent around the crystal does notcrystallize (form ice) but rather freezes in an amorphous glass.Therefore a technique is required to add cryo-protectant to the crystal.

Fifth, many cryo-protectants will damage the crystal. Therefore theremust be an easy way to screen a variety of cryo-protectants.

Sixth, the sudden addition of cryo protectant to a crystal can causedamage to the crystal (e.g. due to osmotic shock). Therefore there mustbe a mechanism for the slow and controlled addition of cryo protectant.

Seventh, when a cryo protectant is added, care must be taken to matchthe relevant chemical variables, including but not limited to saltconcentration, pH, and additive concentrations. If these variableschange appreciably, the crystal contacts may be disturbed, compromisingthe perfection and hence diffracting power of the crystal. In order tomatch these variables precise control over the chemical concentrations,and hence the dehydration, is desired. Therefore a growth device mustallow for exact control over the final state of the reaction.

Eighth, once crystals grow to sufficient size they should be harvestedand frozen as soon as possible to avoid deterioration. Since crystals indifferent wells of a device may appear and grow at different times it isnecessary to be able to harvest single reaction sites from a chipwithout disturbing the others.

Ninth, any background scatter or attenuation of x-ray reflections duringdata collection compromises the quality of the collected data.Therefore, a mounting technique must be arranged that minimizes theinteraction of the x-rays with material that does not contribute to thediffraction signal.

Tenth, to maximize throughput during crystal screening (i.e. todetermine which crystals diffract the best), it should be easy to mountand align crystals in the diffraction beam. Therefore, the crystals mustbe easily visible in the beam with minimal optical aberrations, andmounting more then one crystal at a time is advantageous.

Twelfth, the dead-end filling technique used to fill the previouslydescribed microfluidic devices is slow for large volumes. The harvestingdevice should allow for faster filling.

Embodiments in accordance with the present invention address these andother issues related to obtaining high quality diffraction data usingmicrofluidic devices. In particular, these embodiments make itstraightforward to proceed from initial crystallization conditions(identified using the previously described crystallization chip) to highresolution diffraction data.

FIG. 19A shows a simplified cross-sectional view of an embodiment of a“Microfluidic Protein Crystal Growth and Diffraction Chip” in accordancewith the present invention. Crystal Growth and Diffraction Chip 1700comprises a thick (approximately 1 cm) elastomer layer 1702 with largethrough-holes 1704 (approximately ⅜″ diameter) bonded to a thin(approximately 250 micron) multilayer elastomer 1706 containing flowchannels 1718, microwells 1716, and control channels (not shown in FIG.19A). Thick layer 1702 is aligned so that every reaction centercomprised of 5 pairs of microwells 1716 a-b connected via a microfluidicchannel 1718, is located at the bottom of one through-hole 1704. Theliquid 1705 disposed in through-holes 1720 may be referred to as“osmotic baths” or “vapor diffusion barrier”.

FIGS. 19B-C are micrographs showing perspective and plan views,respectively, of a reaction center at the bottom of an osmotic bath. Thecurrent design of the device has twenty such reaction centers(through-holes). The wells in the reaction centers can have a variety ofrelative volumes, total volumes, and connecting channel geometries.

A cross-section of the membrane separating the osmotic bath from thereaction center, comprising three bonded layers, is shown in FIG. 21.Note that the photograph is inverted, with the bottom of the device atthe top of the picture. The bottom layer is a thin (approximately 20micron) featureless membrane that seals the control channels. The centerlayer is the control layer that contains the control channels. The toplayer is the thickest layer in the membrane (approximately 200 microns)and contains the flow channels (approximately 20 microns high) and thereaction chambers (approximately 150 microns high). The entire membraneis approximately 265 microns thick. Depending upon the particularembodiment, PDMS elastomer material separating crystal forming regionsfrom the adjacent osmotic bath or vapor diffusion barrier, could have athickness as large as 500 μm.

The device of FIGS. 19A-C and 21 is operated as follows. All controllines are first dead-end filled with a fluid (e.g. water or fluorinatedoil). The interface control line that separates the pairs of micro-wellsis actuated and the solutions (protein and precipitant) are dead-endfilled up to the valve. The osmotic baths are filled with differentfluids and are sealed with clear tape. The containment valves areactuated to contain the reactions, and the interface valves are openedto begin the mixing.

When crystals form, they may be harvested by punching out the membraneat the bottom of the osmotic bath. This “crystal-disk” can then be heldin a clip and mounted in the x-ray beam for data collection.

FIG. 55 shows a simplified cross-sectional view of one embodiment of atool which allows the removal of crystal-containing portions of a chipfor analysis. Specifically, tool 2200 comprises piston 2204 sildablewithin enclosure 2206. Piston 2204 comprises handle 2208, ejectorportion 2210 including ejector pin 2214, and blade portion 2211including blade 2212. Tight spring 2216 and loose spring 2218 govern therelative motion of blade portion 2211 and punch portion 2210 relative tothe housing and to one another.

FIG. 56 shows a simplified enlarged view of the tool of FIG. 55 inoperation. Specifically, tool 2200 is positioned over the region of chip2230 containing the crystal 2232 of interest. Next, handle 2208 isbiased in the direction of the chip, compressing loose spring 2218 andallowing blade portion 2211 and ejector portion 2210 of piston 2204 tomove in concert unit within housing 2206, such that blade 2212 engagesand penetrates the elastomer material to separate the crystal-disk fromthe surrounding chip.

Next, the tool is withdrawn from the elastomer, such that the crystaldisk remains held between the blades. Then, in order to free to thecrystal disk from the tool for X-ray diffractometry, handle 2208 iscontinued to be biased forward, such that such that tight spring 2216 iscompressed, and ejector pins 2214 of ejector portion 2211 extend forwardrelative to the now-stationary blade 2212, displacing the crystal diskfor analysis.

FIG. 22 is a micrograph showing a plan view of an extracted crystal diskin accordance with an embodiment of the present invention. FIG. 23 is amicrograph showing a crystal disk in accordance with an embodiment ofthe present invention held in a mounting clip.

An embodiment of a device in accordance with FIGS. 19 and 21-22satisfies the previously listed requirements at least as follows. First,since the mixing is accomplished by diffusion across a microfluidicchannel, the kinetics that were successful in the growth chip arepreserved. These kinetics may be further manipulated by changing thegeometry of the connecting channel as described above. Protein crystalsgrown using the present invention are shown in FIG. 24.

Moreover, since the elastomer is gas permeable, water vapor can easilypass through the thin membrane separating the wells from the osmoticbaths. The wells will therefore come into thermodynamic equilibrium withthe osmotic baths. Since the volume of the osmotic baths is much largerthan that of the wells the final concentration of the solutions in thewells is determined by the osmotic baths. By changing the composition ofthe solutions in each osmotic bath the level of dehydration necessaryfor crystallization may be determined.

Furthermore, since the crystals do not need to be removed from the“crystal disks” there is no need for damaging manipulations.

Once the disk is removed from the chip, it may be directly placed intothe x-ray beam for room temperature data collection. Since the fluid isencased in elastomer, dehydration is slow and data may be collected forseveral hours. If cryo mounting is desired, the crystal disk may besubmerged in liquid nitrogen or some other cryogen. As the disk is verythin, it has low thermal mass and allows for quick freezing of thecrystal.

The addition of cryo-protectant may be accomplished in several ways. Onemethod is to replace the osmotic bath with the cryo-protectant solutionand then slice the membrane over the well connected to the wellcontaining the crystal. This creates a fluidic connection to the crystalwell, allowing the cryo-protectant to diffuse in over time.Alternatively, the membrane over the well containing the crystal couldbe sliced to allow for more rapid addition of cryo-protectant.

Another method of adding cryo-protectant is to remove the disk from thedevice and submerge it in cryo-protectant. This will allow diffusion ofthe cryo-protectant into the wells through the channels open at the edgeof the disk. Alternatively, a separate channel for the addition of thecryo-protectant could be added to the fluidic layer.

Alternatively, in some cases the crystals may grow in the presence of acryo-protectant. Specifically, the elastomer material may be permeableto a cryo-protectant, for example certain types of alcohols. The osmoticbath may comprise such a cryo-protectant.

Where the concentration of this cryo-protectant is sufficient to ensurefreezing of the solution in an amorphous glass, the disks may beextracted and frozen directly. Where the concentration is below thatrequired for formation of an amorphous glass, the cryo-protectant may beconcentrated through vapor diffusion with a concentrate solution in theosmotic bath, thereby obviating any need to perforate the membrane.

Embodiments in accordance with the present invention allow differentcryo-protectant to be screened in each osmotic bath. Moreover, thecryo-protectant may be added very slowly by diffusion as describedabove. In addition, the final concentration of the well solutions isprecisely controlled by the osmotic baths as described above.

While embodiments in accordance with the present invention havedescribed a structure wherein an osmotic bath comprises water, solute,crystallizing agent, or cryo-protectant, the well adjacent to thereaction is need limited to containing one or more of these materials.In accordance with another alternative embodiment of the presentinvention, the elastomer may be permeable to an additive such as across-linking reagent, a ligand, or a small drug molecule.

In accordance with one such alternative embodiment, diffusion of across-linking reagent across the membrane may help to stabilize ancrystal that has formed, for example by forming covalent cross-linksbetween individual proteins within a crystal. The formation of suchcovalent cross-links can result from interaction between functionalgroups including but not limited to amines, thiols, and carboxylicacids. One specific reagent useful in forming such cross-links isglutaraldehyde. A detailed discussion of some approaches for proteincross-linking may be found athttp://www.probes.com/handbook/sections/0502.html, incorporated byreference herein for all purposes.

Embodiments in accordance with the present invention offer the advantagethat the control lines isolating the reaction sites do not intersectwith the edge of the osmotic baths. Therefore, punching out a crystaldisk does not severe the control lines, and therefore does not affectthe other reaction sites.

Moreover, the membrane is very thin to minimize background scatter andattenuation. The X-rays pass through 50 microns of elastomer, followedby 150 microns of solvent and crystal, followed by 50 microns ofelastomer. Thus the scatter and absorption is superior or comparable tostandard techniques.

Furthermore, data may be collected over greater then 120 degrees ofrotation. High resolution diffraction from a protein crystals grown,frozen and harvested using the present invention is shown in FIG. 25.

Since an entire disk is harvested, frozen, and mounted in one step, thistechnique allows for the parallel processing of many crystals, which isnot possible by conventional approaches. And since the wells are only150 microns thick, the chance of two crystals lying on top of oneanother is small. An x-ray beam focused to 100 microns can be used tosequentially interrogate single protein crystals. In this way 100crystals can be screened from a single disk.

Since the elastomer is clear, it is easy to view the crystals for beamalignment. Furthermore, since the disk is very thin, there is negligibleparallax in the case of slight deviation from normal optical incidence,which can pose a problem with thicker devices. Finally, since the gasvents through a very thin membrane during loading, the loading time maybe accelerated, and harvesting may be easily automated.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1-33. (canceled)
 34. A method for mixing a first fluid and a secondfluid in different portions in a microfluidic device, the methodcomprising: providing a microfluidic device comprising a plurality ofcompound wells, wherein each compound well comprises a first well havinga first volume and a second well having a second volume, wherein a ratioof the first volume to the second volume of at least one compound welldiffers from that of the remaining compound wells; filling the firstwells with said first fluid; filling the second wells with said secondfluid; and placing the first well of each compound well in fluidcommunication with the second well, thereby allowing the first fluid andsecond fluid to mix by free-interface diffusion in portions that differbetween compound wells.
 35. The method of claim 34, wherein eachcompound well further comprises a microchannel connecting the first welland the second well, and an interface valve disposed across themicrochannel, and wherein placing the first well of each compound wellin fluid communication comprises releasing the interface valve.
 36. Themethod of claim 34, wherein each compound well further comprises a firstmicrochannel through which said filling of the first well occurs, asecond microchannel through which said filling of the second welloccurs, a first containment valve disposed across the firstmicrochannel, and a second containment valve disposed across the secondmicrochannel, and wherein, before placing the first well of eachcompound well in fluid communication, the first containment valves areactuated after filling the first wells and the second containment valvesare actuated after filling the second wells.
 37. The method of claim 34,wherein filling the first and second wells comprises dead-end filling.38. The method of claim 34, wherein after the first fluid and secondfluid have been allowed to mix, a property of the resulting mixtures ismeasured.
 39. A method of mixing a first fluid and a second fluid inadjustable portions in a microfluidic device, the method comprising:providing a microfluidic device comprising a first well, a plurality offirst control channels disposed across the first well, and a secondwell; filling the first well with said first fluid; filling the secondwell with said second fluid; pressurizing at least one first controlchannel to divide the first well into a plurality of first chambers; andplacing the first well in fluid communication with the second well, suchthat at least one of said first chambers is in fluid communication withthe second well, thereby allowing the first fluid and second fluid tomix by free-interface diffusion in portions determined by thepressurized first control channels.
 40. The method of claim 39, whereinthe microfluidic device further comprises a plurality of second controlchannels disposed across the second well, and said method furthercomprises pressurizing at least one second control channel after fillingthe second well, thereby dividing the second well into a plurality ofsecond chambers.
 41. The method of claim 39, wherein the microfluidicdevice further comprises a microchannel connecting the first well andthe second well; and an interface valve disposed across themicrochannel, and wherein placing the first well in fluid communicationwith the second well occurs by releasing the interface valve.
 42. Themethod of claim 39, wherein the microfluidic device further comprises afirst microchannel through which said filling of the first well occurs,a second microchannel through which said filling of the second welloccurs, a first containment valve disposed across the firstmicrochannel, and a second containment valve disposed across the secondmicrochannel, and wherein, prior to placing the first well in fluidcommunication with the second well, the first containment valve isactuated after filling the first well and the second containment valveis actuated after filling the second well.
 43. The method of claim 39,wherein filling the first and second wells comprises dead-end filling.44. The method of claim 39, wherein after the first fluid and secondfluid have been allowed to mix, a property of the resulting mixtures ismeasured.
 45. A method of mixing a first fluid and a second fluid atvarying rates in a microfluidic device, the method comprising: providinga microfluidic device comprising a plurality of compound wells, whereineach compound well comprises a first well, a second well, and one ormore microchannels connecting the first and second wells, wherein theamount, average length, and/or average cross sectional area of themicrochannels vary between compound wells; filling the first wells withsaid first fluid; filling the second wells with said second fluid; andplacing the first wells in fluid communication with the second wells viathe microchannels, thereby allowing the first fluid and second fluid tomix by free-interface diffusion at rates that vary between compoundwells.
 46. The method of claim 45, wherein each compound well in saidmicrofluidic device further comprises at least one interface valve, suchthat one interface valve is disposed across each microchannel, and saidplacing step occurs by releasing the interface valves.
 47. The method ofclaim 45, wherein each compound well in said microfluidic device furthercomprises: a first filling microchannel through which said filling ofthe first well occurs, a second filling microchannel through which saidfilling of the second well occurs, a first containment valve disposedacross the first filling microchannel, and a second containment valvedisposed across the second filling microchannel, and wherein, prior toplacing the first wells in fluid communication with the second wells,the first containment valves are actuated after filling the first wells,and the second containment valves are actuated after filling the secondwells.
 48. The method of claim 45, wherein filling the first and secondwells comprises dead-end filling.
 49. The method of claim 45, whereinafter the first fluid and second fluid have been allowed to mix, aproperty of the resulting mixtures is measured.
 50. A method ofcapturing, in a microfluidic device, a chemical gradient resulting fromthe diffusive mixing of a first fluid and a second fluid, the methodcomprising: providing a microfluidic device comprising a first well, atleast one first control channel disposed across the first well, and asecond well; filling the first well with said first fluid; filling thesecond well with said second fluid; placing the first well in fluidcommunication with the second well, thereby allowing the first fluid andsecond fluid to begin mixing by free-interface diffusion; andpressurizing at least one first control channel before mixing hascompleted, thereby dividing the first well into a plurality of firstchambers, such that concentrations of the first fluid and second fluiddiffer between first chambers.
 51. The method of claim 50, wherein themicrofluidic device further comprises at least one second controlchannel disposed across the second well, and wherein at least one secondcontrol channel is pressurized after placing the first well in fluidcommunication with the second well during free-interface diffusion,thereby dividing the second well into a plurality of second chamberssuch that concentrations of the first fluid and second fluid differbetween second chambers.
 52. The method of claim 50, wherein themicrofluidic device further comprises a microchannel connecting thefirst well and the second well, and an interface valve disposed acrossthe microchannel, and wherein placing the first well in fluidcommunication with the second well occurs by releasing the interfacevalve.
 53. The method of claim 50, wherein the microfluidic devicefurther comprises a first microchannel through which said filling of thefirst well occurs, a second microchannel through which said filling ofthe second well occurs, a first containment valve disposed across thefirst microchannel, and a second containment valve disposed across thesecond microchannel, and wherein, prior to placing the first well influid communication with the second well, the first containment valve isactuated after filling the first well and the second containment valveis actuated after filling the second well.
 54. The method of claim 50,wherein filling the first and second wells comprises dead-end filling.55. The method of claim 50, wherein after the first fluid and secondfluid have been allowed to mix, a property of the resulting mixtures ismeasured.